Airway wall remodeling in asthma : novel mechanisms of human bronchial smooth muscle cells in the induction of angiogenesis

Asthma is a global major health concern and it affects estimated 300 million people. The prevalence of asthma is rising and there is no cure for asthma, only the symptoms can be controlled. Acute asthma attacks are characterized by severe symptoms such as breathlessness, wheezing, tightness of the chest, and coughing, which may lead to hospitalization or death. Besides the acute symptoms, asthma is characterized by persistent airway inflammation and airway wall remodeling. The term airway wall remodeling summarizes the structural changes in the airway wall: epithelial cell shedding, goblet cell hyperplasia, hyperplasia and hypertrophy of the bronchial smooth muscle (BSM) bundles, basement membrane thickening and increased vascular density. Airway wall remodeling starts early in the pathogenesis of asthma and today it is suggested that remodeling is a prerequisite for other asthma pathologies. Furthermore, novel invasive therapies used to treat severe asthma provide evidence that the BSMC is a major effector cell in the pathology of asthma. In the present thesis novel mechanisms of BSMC regulation and their role in the induction of asthma-associated angiogenesis have been elucidated. Therefore, the differences in the angiogenic capacities of BSMC from asthma and non-asthma patients and their modification by different conditions, such as an (i) inflammatory microenvironment, (ii) the influence of low oxygen concentration, and (iii) the stimulation with the most potent asthma relevant allergen (house dust mite (HDM) allergen) on the angiogenic properties of BSMC have been studied. A major finding of this thesis is the increased angiogenic potential of BSMC from asthma patients and the altered release of CXCR2 ligands in an in vitro inflammatory environment. It has been demonstrated that BSMC from asthma patients release significantly more of the CXCR2 ligands ENA-78, GRO-a and IL-8, which may explain the increased vascular density in the sub-epithelial cell layers observed in the airways of asthma patients. These finding adds to previous studies showing that BSMC are a source of angiogenic factors (e.g. VEGF) and that CXCR2 ligands are elevated in the airway lining fluids of asthma patients. In this thesis BSMC have been shown to be a potential source of CXCR2 ligands, which induced spout outgrowth from endothelial cell spheroids in an in vitro model of angiogenesis. Furthermore, this thesis investigated the effect of hypoxia on BSMC. Local restricted hypoxia in the airways of asthma patients had only recently been suggested. The animal model based hypothesis that hypoxia directly causes BSMC hyperplasia was tested. This hypothesis was not confirmed in human BSMC but nonetheless it was demonstrated that hypoxia leads to increased release of inflammatory and angiogenic mediators; as conditioned medium from BSMC grown under hypoxia induced angiogenesis in an in vitro model via VEGF. These findings suggest that different conditions or stimuli induce angiogenesis in asthma through different pathways and therefore, different therapeutic strategies might be needed. In the third part of this thesis the effect of HDM allergen on the release of inflammatory and angiogenic mediators from BSMC was assessed. Animal models demonstrated that exposure to HDM allergens increased airway wall vascularization. No direct contribution of BSMC to HDM extract induced angiogenesis was observed. However, HDM extract proteases degraded ENA-78, which is an import chemokine for neutrophil recruitment into the inflamed lung. Thus HDM allergens may alter the bio-availability of ENA-78 in the airways of asthma patients and modulate the immune response. The findings of this thesis add a small piece to the knowledge of asthma pathology, the mechanisms underlying airway wall remodeling and in particular BSMC hyperplasia and neovascularization. This might represent novel targets for treatment, especially for the prevention or reversal of airway wall remodeling

Proteolytic Activity Present in House-Dust-Mite Extracts Degrades ENA-78/CXCL5 and Reduces Neutrophil Migration

Background. Bronchial smooth muscle cells (BSMC) are a major source of proinflammatory and proangiogenic cytokines and chemokines, including VEGF and CXC-chemokines. CXC-chemokines act primarily on neutrophils, mediating their recruitment to and activation at the site of inflammation. In humans, house-dust mite (HDM) allergens can cause asthmatic exacerbations and trigger an inflammatory response through protease-dependent mechanisms. Objective. We investigated the effect HDM extract on the release of pro-angiogenic and proinflammatory cytokines from BSMC. Methods. Human primary BSMC were stimulated with HDM extract in the absence or presence of fetal calf serum (FCS). Twenty angiogenic cytokines were detected by a specific antibody array and modified protein levels were confirmed by ELISA. Neutrophil migration was measured using a 96-well Boyden chamber. Results. ENA-78/CXCL5 protein levels in conditioned medium of BSMC stimulated with HDM extract were significantly reduced (n=10, P<0.05) but restored in the presence of 5% FCS. HDM extracts did not affect ENA-78/CXCL5 mRNA levels. Recombinant ENA-78/CXCL5 was degraded after incubation with HDM extracts (n=7, P<0.05) but restored after the addition of the serine protease AEBSF. Neutrophil migration towards recombinant ENA-78/CXCL5 was also reduced in the presence of HDM extract. Conclusion. HDM proteases degrade ENA-78/CXCL5. Thus exposure to HDM allergens may alter ENA-78/CXCL5 levels in the lungs and may affect angiogenesis and the inflammatory response in the airways of asthma patients

Western blot analysis detecting HIF-1α and GAPDH (loading control) in lysates of BSMC.

<p>Cells were incubated under 1% and 21% O₂ for 4, 24 and 48 h (indicated at top). CoCl₂ was used as positive control to stabilize HIF-1α protein. In contrast to CoCl₂, which has a transient effect only, hypoxia induced a prolonged expression of HIF-1α. The experiments shown are representative for 5 independent experiments. The right hand panel (showing the 24 h and 48 h expression levels of HIF-1α) was obtained after longer exposure of the same blot, which contained all samples.</p

Proliferation characteristics of BSMC from asthmatic (A) and non-asthmatic (NA) subjects in the presence of 1%, 5% and 21% O₂ (72 h).

<p>BSMC were cultured for 72 hours in presence (<b>A</b>) and absence (<b>B</b>) of 5% FCS. The data presented in <b>A</b> and <b>B</b> are shown as one group (6A+6NA for 21% O2 and 4A+3NA for 1% and 5% O₂), as well as two separate groups (6A and 6NA for 21% O_2; 4A and 3NA for 1% and 5% O2). <b>C</b> and <b>D,</b> densitometric analysis of PCNA (9 independent experiments in 5 subjects) and cyclin E (6 independent experiments in 5 subjects) under hypoxic (1% O₂) and normoxic conditions (21% O₂). <b>E</b> and <b>F,</b> representative Western blots for PCNA and cyclin E protein expression. Densitometric values are given as mean ± SEM (*p-value ≤0.05, **p-value ≤0.01, ***p-value ≤0.001).</p

Light microscopic photographic images of airway tissue sections obtained from a non-asthmatic (A) and an asthmatic (B) patient.

<p>Images (magnification 60×) are representative of tissues obtained from 3 non-asthmatic and 7 asthmatic patients stained with Haematoxylin-Eosin. Note the increased thickening of the basement membrane in the asthmatic airways. E = epithelium, arrows are indicating the basement membrane.</p

Normoxia (21% O2) versus hypoxia (1% O2). Effects on cytokine release from BSMC grown in the presence of 5%FCS (72 h).

<p>Concentrations of VEGF (<b>A</b>), IL-6 (<b>B</b>) and IL-8 (<b>C</b>) in CM collected from BSMC of 5 non-asthmatic and 5 asthmatic subjects were determined by ELISA. Values are given as mean ± SEM. *p-value ≤0.05, *p-value ≤0.01, ***p-value ≤0.001 (all relative to 21% O₂). Abbreviations: A = asthmatics, NA = non-asthmatics.”</p

Pathomechanistic characterization of two exonic L1CAM variants located in trans in an obligate carrier of X-linked hydrocephalus.

International audienceMutations in the gene encoding the neural cell adhesion molecule L1CAM cause several neurological disorders collectively referred to as L1 syndrome. We report here a family case of X-linked hydrocephalus in which an obligate female carrier has two exonic L1CAM missense mutations in trans substituting amino acids in the first (p.W635C) or second (p.V768I) fibronectin-type III domains. We performed various biochemical and cell biological in vitro assays to evaluate the pathogenicity of these variants. Mutant L1-W635C protein accumulates in the endoplasmic reticulum (ER), is not transported into axons, and fails to promote L1CAM-mediated cell-cell adhesion as well as neurite growth. Immunoprecipitation experiments show that L1-W635C associates with the molecular ER chaperone calnexin and is modified by poly-ubiquitination. The mutant L1-V768I protein localizes at the cell surface, is not retained in the ER, and promotes neurite growth similar to wild-type L1CAM. However, the p.V768I mutation impairs L1CAM-mediated cell-cell adhesion albeit less severe than L1-W635C. These data indicate that p.W635C is a novel loss-of-function L1 syndrome mutation. The p.V768I mutation may represent a non-pathogenic variant or a variant associated with low penetrance. The poly-ubiquitination of L1-W635C and its association with the ER chaperone calnexin provide further insights into the molecular mechanisms underlying defective cell surface trafficking of L1CAM in L1 syndrome

Involvement of CXCR2 in BSMC-induced neovascularization.

<p>A, RT-PCR of CXCR2 in HMEC-1. B, Immunofluorescence detection of CXCR2 on HMEC-1. C, D, HMEC-1 monolayers under normal EC growth conditions were cultured without or with SB 265610 for 48h (C) or 24h (D) (mean ± SD, n=3) and evaluated for proliferation (C) and viability (D). E, Effect of SB 265610 on sprout outgrowth induced by CM of BSMC from asthmatics. Values are mean ± SEM after normalization to the control condition. *** p < 0.001 (n ≥ 3).</p

Human angiogenesis antibody array.

<p>Examples of the angiogenesis antibody array (exp. 3) comparing CM from BSMC of non-asthmatic (A) and asthmatic (B) patients. C, Antibody array map. Standard abbreviations for the detected proteins are used, Pos: positive control, Neg: negative control, IC1-IC3: internal controls 1-3. D, Quantitative analysis from each of the 4 independent experiments performed. Intensity ratios (A:NA) in a paired analysis are shown. Upregulation in any single experiment is indicated by a cross. nq = not quantifiable (out of range).</p

Milligan’s trichrome stained sections of airway tissue from non-asthmatic (A) and asthmatic (B) patients.

<p>Images are representative of tissues obtained from 3 non-asthmatic and 3 asthmatic patients. Nuclei and muscle: magenta, collagen: green, RBC: orange. Note epithelial hyperplasia, thickening of muscle bundles and basement membrane, and increased micro-vessel density in asthmatic airways. E = epithelium, MB = muscle bundles, BM = basement membrane, MV = micro-vessels. </p