RICERCA ITALIANA     

New insights of inflammation and remodeling in airway obstructive deseases

Università degli Studi di Genova

Abstract

There are multiple cellular and molecular pathways leading to inflammation, remodelling and chronicization in obstructive airways diseases. The Th2-type inflammation with eosinophilia existing in some disorders without obstruction (e.g. persistent rhinitis and eosinophilic bronchitis) is a good starting point to investigate novel mechanism of inflammation. Recently, some experimental models of inflammation-remodelling, involving the intrinsic properties of the smooth muscle cells and independent of the Th2 inflammation, have been proposed. Among these mechanisms are: rhinoviral infections, bradikinin, vascular remodelling, desensitization of beta2 receptors. All these phenomena, alone or in combination, may contribute to those structural and functional changes in smooth muscle, which result in increased bronchial responsiveness and reduced bronchial calibre.
These coordinated projects have the aim of investigating the following aspects:

- role of acethylcholine, bradikinin and VEGF in contractile remodelling of smooth muscle cell;
- desensitizing effect of leukotrienes and thromboxanes on Beta2 receptors;
- effects on the release of acethylcholine from bovine smooth muscle of INFgamma and lambda and of the supernatant of rhinovirus-infected epithelial cells;
- signal trasduction of cysLT and TP receptors and mechanims regulating the desensitization of beta2 receptors;
- production of interferons and acethylcholine by rhinovirus-infected epithelial cells;
- role of bradikinin in inducing or regulating the fibroblast-myofibroblast remodelling and VEGF production;
- VEGF expression and vascular permeability in asthma.

The unit coordinated by Canonica will assess the remodelling effect of acethylcholine, bradykinin and VEGF on cultured smooth bronchial muscle. This will be made by studying the contractile proteins and the cellular stiffness by means of magnetic twinsting cytometry. Utilizing the passive sensitization, the unit will also assess the role of leukotrienes, bradykinin and thromboxanes in desenzitizing the beta2 adrenoceptors. In cooperation with Papi, using a quantitative assay for acethylcholine release from bronchial bovine tissue, the effects of supernatant of rhinovirus infected cells and interferons will be assayed. In the unit coordinated by Papi, cultured epithelial cells will be infected with rhinoviruses and the production of interferons (expecially lambda interferon) and acethylcholine will be studied. The unit coordinated by Rovati will complete the characterization of the signal transduction pathways of CysLT1 and TP receptors. In cooperation with Canonica the enzymatic pathways involving the phosphorilation of Beta2-Gs receptors will be studied. The units coordinated by Vancheri and Chetta will define in vitro and in vivo the mechanisms of remodelling induced by bradykinin and VEGF, and the regulatory effect of bradikinin on VEGF production. The results from these latter units will be integrated with the experiments of the Canonica's unit on the pro-contractile remodelling caused by bradykinin and VEGF. <<<

Principal Investigator

Giorgio Walter CANONICA Università degli Studi di GENOVA

Research Objectives

The present research programme has the general aim of investigating some aspects of bronchial inflammation and remodelling in obstructive diseases that are poorly known, yet suspected to play a crucial role.
The following aspects will be considered:
- role of acethylcholine, bradykinin and vascular endothelial growth factor (VEGF) in the contractile remodelling of human airway smooth muscle cells;
- signal trasduction by leukotrienes (cysLT) and thromboxane (TP) receptors and mechanisms that regulate the eterologous desensitization ob beta2 receptors;
- effects of rhinoviral infection of epithelial cells in regulating the production of interferons and acethylcholine;
- role of bradykinin in the process leading to fibroblast-myofibroblast remodelling, remodelling of the human airway smooth muscle cell and production of VEGF;
- VEGF expression and vascular permeability in asthma.

The research project involves 5 interdependent Units.

Canonica: To assess the effects of acethylcholine, BK and VEGF in remodelling the human airway smooth muscle cells; in cooperation with Rovati and Vancheri, to assess the effects of thromboxanes and leukotrienes in eterologous desenzitization of the beta2 receptor; in cooperation with Papi, to evaluate the effect of interferons and supernatant from rhinovirus infected epithelial cells on acethylcholine release from bovine bronchial tissue; in cooperation with Chetta to study fibronectin production from cultured human airway smooth muscle cells stimulated with VEGF.

Rovati. To complete the characterization of the signal transduction pathways of the Cys-LT and TP, as they are involved in the remodelling process; to define the possible relationship existing between Cys-LT and TP and the function of the beta2 receptor in human airway smooth muscle. Main steps will be: 1) detailing the signalling transduction of CysLT and TP with a particular attention to PI3K and PKC pathways; 2) characterization of the genes involved; 3) assessment of the role of CysLT and TP in the desensitization of beta2 receptor. This latter part will be shared with Canonica.

Vancheri: to understand the mechanims and the factors that lead to the transformation of fibroblasts into myofibroblasts; to assess the role of bradikinin in airways remodelling; to detail the cellular and molecular events leading to fibroblast transformation; to assess the role played by bradikinin in VEGF-mediated angiogenesis.

Papi. To evaluate the mechanisms intervening in viral infections, with special attention to the production of lambda inteferon; to assess which are the differences between asthma patients and healthy subjects in response to rhinoviral infection; to study the effects of rhinoviral infection (in cultured epithelial bronchial cells) on the production of choline acethyltransferase that is involved in acethylcholine synthesis; to assess the relationship between antiviral response and bronchial hyperreactivity. This latter point would define a mechanism that justifies the transient increased hyperreactivity during viral infeactions in asthma. The underlying hypothesis is that in asthma patients the replication of rhinovirus is increased, and that the increased single stranded RNA induces the release of TH1 cytokines. Among these cytokines, interferons could down regulate the M2 receptor (tested by Canonica et al.) and/or upregulate the Cys-LT receptors. Moreover an increased infectivity of rhinoviruses may be related to an increased production of acethylcholine in bronchial epithelium.

Chetta: To understand the role of VEGF in bronchial wall remodelling, angiogenesis and bronchial reactivity in obstructive airways disease (asthma, COPD); to verify if in the airways of asthmatic or COPD subjects, an increased production of VEGF is associated to increased vascularity and subepithelial fibrosis. To test the working hypothesis, the epithelial expression of VEGF will be compared among asthmatics, COPD patients and healthy volunteers. To assess in both COPD and asthma patients, the cough reflex and its correlation with the degree of bronchial remodelling. <<<

Timescale

24 months

National and international background

The "remodelling" is defined as a chronic alteration of the cellular and molecular components of the bronchial airways (1). Indeed there are multiple and redundant pathogenic mechanisms leading to both inflammation and remodelling.
Asthma is characterized by a global remodelling of the bronchial wall. The bronchial epithelium is thinner than in normals, repairing processes are slow and polarized towards the secretion of growth factors. Moreover, the vagal activity is increased and there is an increased release of acetylcholine. Mucinous glands are hyperthrophic and secrete large amount of mucus (2). In asthma, but not in COPD, the subepithelial connective layer is thicker than in normals, and a similar finding is present in subjects with perennial rhinitis but not asthma (3). It has been shown that an inverse correlation exists between subepithelial thickening and bronchial hyperresponsiveness (4,5). This suggest that subepithelial changes may act as a protective factor that limits the maximal bronchoconstriction achievable. In submucosa, there is an abnormal activation of fibroblasts, myofibroblast are present (6) and an excess of deposition of intercellular matrix, associated with neoangiogenesis.
This latter aspect is present also in COPD, but not in eosinophilic bronchitis. A central role seems to be played by the Vascular Endotehelial Growth Factor (VEGF) that has a broad spectrum of activities including angiogenesis and increase of vascular permeability. The most recent studies showed that VEGF is increased in asthma, but not in eosinophilic bronchitis (7).
Concerning the smooth muscle, it is hyperthtrophic in both asthma and COPD, but in this latter diseases it seem to be confined to the most distal airways. Smooth muscle cells are largely responsible for the changes in bronchial calibre. Thus, structural changes in smooth muscle cells are thought to contribute significantly to the functional alterations in asthma and COPD.
The present interuniversity coordinated project has the main aim of assessing some novel, and so far not deeply investigated, aspects of remodelling and inflammation in obstructive lung diseases. The main research directions will be the following:
- role of acethylcholine, bradikinins and VEGF in the remodelling processes involving human cultured smooth muscle cells;
- desensitizing effect of leukotriene, thromboxanes and bradykinin on beta2 receptors, studied through the experimental model of ex-vivo passive sensitization;
- acethylcholine release from bovine bronchial smooth muscle, challenged with interferons and supernatant from rhinovirus-infected epithelial cultures
- signal transduction from the leukotriene (CysLT1) and thromboxanes (TP) receptors and mechanisms of eterologous desensitization of airway smooth muscle Beta2 receptors
- effects of rhinoviral infections on epithelial cells and production of acethylcholine and interferons
- role of bradykinin in epithelial and fibroblastic remodelling and modulation of VEGF release
- expression of VEGF in bronchial biopsies from asthmatic subjects and its relationship with increased permeability and hyperreactivity.
The interrelationships and connections among the projects and cooperations are explained and detailed in the chapters below. The name(s) of the project's responsible(s) in parentheses indicate the single or multiple involvement of groups in each project.


Inflammation in obstructive lung diseases may be sustained by different cell types, namely eosinophils, mast cells, lymphocytes, neutrophils. These cells release a variety of preformed or neo-synthesized mediators that can stimulate the receptors on the surface of smooth muscle cells, thus leading to various effects. The effect of a given mediator depends on the activation of a G-coupled receptor, that activates specific enzimatic cascades. In this context a given mediator may cause both a mechanical and a synthetic/proliferative response. Also acethylcholine can be considered an inflammatory mediator. It is released by vagal nerve ends following irritative stimuli, but it is also present in mast cells, macrophages, neutrophils and myocites. All these cells can store acethylcholine as preformed mediator or syntehsize it through the enzyme acethyl-transferase. It is reasonable to hypothesize that acethylcholine, besides the neuromodulatory effect, can also modulate the phenotypic characteristics of smooth muscles (8). Its main excitatory transduction pathway involves the M3 receptor coupled with a Gq protein and C-phospholipase, whereas the inhibitory pathway involves the M2 receptor coupled with Gi protein, and blocks the synthesis of cAMP. This organization of the receptor activities leads to contemporary contraction and inhibition of relaxation of the muscle cell. M2 receptors are largely represented on smooth muscle cells and vagal nerve ends. Their main task is to inhibit acethylcholine release via negative feedback. A disruption of this control mechanism can be provoked, for instance, by the eosinophil major basic protein released during allergic inflammation, by neuroaminidase during viral infections, by ozone, and also by vitamin A deficiency (9). Moreover, some of the mediators contained in smooth muscle cells (namely thromboxane A2 or Prostaglandin D and F2alfa) can enhance the release of acethylcholine (9). Such as for other mediators acting by the G-coupled receptors, also acethylcholine can activate a protein kinase C (PKC) isoform. This isoform, is in turn, capable of activate (via Raf 1) the p42/p44 Mitogen Activated kinases (MAP), as demonstrated by the selective blockage of PKC by the compound G109203X (10). In parallel, the M2-Gi pathway can activate the p42/p44 MAP kinase by an alfa-i dependent pathway through Ras, or a beta/gamma-i dependent or Rho dependent phosphatidil inositol 3 kinases (P13K)activation and, again, by transactivation of tyrosin-kinase receptors.
In general, the proliferative phenotype is considered to be mutually exclusive with respect to the contractile one, nevertheless, some growth signals can lead to pro-contractile remodelling phenomena. The effects of acethylcholine on muscle cell phenotype are complex and still controversial, since muscarinic receptors can both enhance or reduce the contractility of smooth muscles. In fact, stimulation with acethylcholine increases intracellular calcium, which decreases contractility (11). On the other hand in dog smooth muscle cells, stimulation with carbachol results in increased myosin and SM22/transgelin synthesis via Rho- and Rho-kinase (12). Baseline phenotype of smooth muscle cells may greatly condition the experimental results. The greater gene transcription has been observed with synthetic phenotypes (13). The smooth muscle cell can be oriented towards a contractile phenotype after a maturational arrest induced by serum deprivation or in the presence of insulin (14). Insulin seems to act via down regulation of the M2 receptor. Contractile phenotype is characterized by a greater contraction velocity and enhanced expression of proteins such as actin, myosin and myosin light chain chinase. Moreover, in these conditions, also the M3 receptor expression is increased (15). The complexity of the acethylcholine-induced remodelling requires therefore an approach allowing to assess at the same time structural and functional changes. This can be done by means of the magnetic twisting cytometry (MTC) which allows to quantitatively study smooth muscle cell contractility by its stiffness. For details see (Canonica) project.

Characterization of signal transduction of CysLT1 and TP receptors activation is far from being completed. Leukotrienes are potent (1,000 times than histamine) bronchoconstrictor stimuli, and their action is not strictly associated to an increase of intracellular calcium. Thus, a pathway different from that involving phospholipase C is conceivable. It is likely that the response to leukotrienes is dependent on the PKCepsilon concerning both calcium-induced and heterologous beta2 sensitization (16). Other pro-contractile mediators (prostaglandin D2, thromboxane A2) have receptors (TP) that are preferably Gq coupled. A recent study (17) has shown that stimulating those receptors induces the proliferation of smooth muscle cells, independent of activation of leukotriene receptors. It is not known if activation of TP receptors can affect the contractility of the smooth muscle or is able to desensitize Beta2 receptor. (Rovati-Canonica)
The Beta2 adrenoceptors (B2AR) can be desensitized by some protein kinases (PKC or PKA), activated in turn by a receptor that is different from the B2AR. In the case of asthma, a chronic hyperstimulation of the CysLT1 or TP receptors, due to enhanced production of leukotrienes and thromboxanes, may lead to a desensitization of the B2AR, with decreased efficacy of the B2 bronchodilators. (Canonica-Rovati).
Upper respiratory viral infections are known causes of asthma and COPD exacerbation. These infections usually elicit, in normal individuals, a Th1 response with production of large amounts of interferons. This response may be altered in asthmatic patients. The production of interferons induces an anti-viral protection by the secretion of proteins that interfere with viral replication, by enhancing the activity of NK cells and by increasing MHC-class1 antigen expression (18). Very recently, a new class of interferons, called lambda-interferons, has been described (19). The exact functions of lambda interferons are still unknown. It is known that interferon gamma increase the expression of CysLT1 receptors (20). Thus, the increased bronchial responsiveness observed in predisposed subjects following a respiratory infection might be explained by the increased production of interferons (21) (Papi).
When comparing the activity of leukotriene D4 and bradikynin on smooth muscle cells, it has been observed that gamma interferon enhances the effect of leukotrienes, whereas bradikinin is quite independent of the presence of interferon in producing increase of smooth muscle stiffness(20) (Canonica). In addition, gamma interferon increases the release of acethylcholine, by down-regulating M2 receptors, and this fact may contribute to the increase in bronchial hyperreactivity that follows viral infections. (Papi-Canonica).
In ex vivo models of passive sensitization and allergic inflammation, it was suggested that the Beta2-Gs complex desensitization is strictly associated with leukotrienes, since premedication with montelukast, a leukotriene antagonist, blocks the desensitization of B2AR (22). It has been suggested that PKC itself can be involved causing phosphorilation and uncoupling of receptor from Gs protein. Thus, either in Th1 driven viral infection or in Th2 allergic inflammation, leukotrienes may play a central role in modulating the contraction of smooth muscles (Rovati-Papi-Canonica).
Bradykinin (BK) is a mediator with potent vasodilator and bronchoconstrictor action. It is a good candidate as key mediator in asthma since: a) bradikinin is present at high concentration in the upper airways of asthmatic subjects after allergen exposure (23); b) specific antagonists of the bradikinin receptors inhibit the increase of inflammatory mediators in the airways and decrease the bronchial hyperresponsiveness(24). BK is also involved in the pathogenesis of viral-induced bronchial hyperresponsiveness and in neurogenic inflammation and cough (25). Recently it has been shown that the production of VEGF by smooth muscle is promoted by BK (26). Its role in promoting the differentiation from fibroblast into myofibroblast and in the development of a contractile phenotype in airway smooth muscle remodeling is not well known (Vancheri-Canonica).
VEGF is thought to play a key role in the vascular remodelling. Patients with asthma display increased expression of VEGF as compared to healthy individuals and patients with eosinophilic bronchitis (27,7). Macrophages, eosinophils and CD34+ cells are important sources of the mediator. Moreover, it has been shown that VEGF induces fibronectin secretion by myocites in a ERK-dependent way (28). The smooth muscle cell itself is able to synthesize and secrete VEGF, and this secretion is potentiated by BK (29). The presence and role of VEGF in different asthma severity, its correlations with subepithelial fibrosis, and its action on fibroblasts and smooth myocites deserve further studies (Chetta-Vancheri-Canonica). <<<