Abstract Rationale Airway remodelling (AR) contributes to airflow limitation and poor symptom control in severe asthma. While anti-IL-5 therapy improves clinical outcomes in severe asthma, the cellular and molecular mechanisms underlying its effects on AR remain incompletely understood. Objectives. To determine whether IL-5 blockade directly modulates airway epithelial biology and contributes to attenuation of AR in severe asthma with eosinophilia (SAE). Methods. Patients with SAE underwent bronchoscopy before and after 24 weeks of anti-IL-5 therapy. Paired bronchial brushings (n=12) were analysed using single-cell RNA-sequencing. Histological features of AR were assessed in paired bronchial biopsies (n=16). Functional effects of IL-5 were investigated using wound healing assays in differentiated air-liquid interface (ALI) cultures. Measurements and Main Results. Anti-IL-5 treatment improved clinical outcomes without altering airway epithelial cellular composition. Differential gene expression was predominantly restricted to bronchial ciliated epithelial cells, which expressed IL5RA . ALI cultures showed IL-5Rα protein. Anti-IL-5 therapy induced a transcriptional signature in ciliated cells that opposed IL-5-responsive genes. Pseudotime analyses demonstrated preserved epithelial differentiation trajectories but altered programmes related to mucus regulation and ion transport. Cell-cell communication analyses revealed decreased T2-inflammatory processes alongside enrichment of epithelial repair and barrier integrity processes after treatment. Functionally, IL-5 directly impaired epithelial wound repair in ALI cultures. Histological assessment demonstrated increased epithelial E-cadherin expression and reduced sub-basement membrane thickness, extracellular matrix deposition and goblet cell hyperplasia in bronchial biopsies. Conclusions. IL-5 blockade modulates epithelial biology at transcriptional, functional and structural levels in SAE and is associated with improved epithelial integrity and reduced features of AR. Impact Our findings broaden current understanding of interleukin (IL)-5 biology by demonstrating that IL-5 blockade exerts direct effects on the bronchial epithelium in addition to its established effects on eosinophilic inflammation in asthma. Through single-cell analyses of human bronchial samples, complemented by functional and histological validation we provide novel mechanistic insight into how IL-5 blockade influences epithelial repair, barrier integrity and airway remodelling. These findings advance understanding of severe asthma pathogenesis, provide evidence that structural disease is modifiable and may inform future approaches to disease modification. At a Glance Commentary Scientific knowledge on the subject Interleukin-5 (IL-5) is a key driver of eosinophilic inflammation in severe asthma and anti-IL-5 therapies improve clinical outcomes. Emerging evidence suggests that IL-5 blockade may also improve features of airway remodelling. However, the cellular mechanisms underlying airway structural improvements and the effects of IL-5 blockade on airway structural cells remain poorly understood. What this study adds to the field Using paired bronchial samples obtained before and after anti-IL5 treatment, we demonstrate that IL-5 blockade directly modulates bronchial epithelial cell biology. We identify ciliated epithelial cells as an IL-5 responsive population and show that anti-IL-5 therapy alters transcriptional programmes in airway epithelium associated with repair, barrier integrity and airway remodelling. IL-5 blockade shifts cell-cell communication away from inflammation pathways towards pathways supporting epithelial repair. Functionally, IL-5 directly impairs wound healing of differentiated airway epithelial cells in vitro and IL-5 blockade enhances epithelial integrity and reduces remodelling-associated structural changes in vivo . Our findings broaden current understanding of IL-5 biology by demonstrating that the effects of IL-5 blockade extend beyond eosinophil suppression to include direct effects on biological pathways in epithelial cells involved in repair, barrier integrity and airway remodelling in severe asthma.

SARS-CoV-2 respiratory-tract infection affects both vaccinated and unvaccinated persons suggesting factors besides adaptive immunity are operative. We investigated the role of collectin-11 (CL-11), an epithelial-secreted carbohydrate-binding lectin that drives innate immunity and eliminates pathogens by complement activation. SARS-CoV-2, despite binding CL-11 to activate complement, was resistant to lysis. Remarkably, opsonization by CL-11 enhanced virus production by infected respiratory epithelial cells independently of complement. Furthermore, infected cells expressing SARS-CoV-2 spike protein displayed enhanced vulnerability to CL-11 binding and membrane attack by complement. The mechanism of enhanced infectivity was ablated in the presence of L-fucose, which occupied the extended carbohydrate-binding cleft of CL-11 in a crystallographic analysis of complexes between L-fucose and CL-11. Our study suggests pathogenicity of SARS-CoV-2 is related to complement-resistance together with enhanced infectivity and injury of respiratory epithelial cells mediated by locally released CL-11.

Introduction: Asthma is the most common chronic inflammatory disease of the airways. The airway epithelium is a key driver of the disease, and numerous studies have established genome-wide differences in mRNA expression between health and asthma. However, the underlying molecular mechanisms for such differences remain poorly understood. The human TTP family is comprised of ZFP36, ZFP36L1 and ZFP36L2, and has essential roles in immune regulation by determining the stability and translation of myriad mRNAs encoding for inflammatory mediators. We investigated the expression and possible role of the tristetraprolin (TTP) family of RNA binding proteins (RBPs), poorly understood in asthma.Methods: We analysed the levels of ZFP36, ZFP36L1 and ZFP36L2 mRNA in several publicly available asthma datasets, including single cell RNA-sequencing. We also interrogated the expression of known targets of these RBPs in asthma. We assessed the lung mRNA expression and cellular localization of Zfp36l1 and Zfp36l2 in precision cut lung slices in murine asthma models. Finally, we determined the expression in airway epithelium of ZFP36L1 and ZFP36L2 in human bronchial biopsies and performed rescue experiments in primary bronchial epithelium from patients with severe asthma.Results: We found ZFP36L1 and ZFP36L2 mRNA levels significantly downregulated in the airway epithelium of patients with very severe asthma in different cohorts (5 healthy vs. 8 severe asthma; 36 moderate asthma vs. 37 severe asthma on inhaled steroids vs. 26 severe asthma on oral corticoids). Integrating several datasets allowed us to infer that mRNAs potentially targeted by these RBPs are increased in severe asthma. Zfp36l1 was downregulated in the lung of a mouse model of asthma, and immunostaining of ex vivo lung slices with a dual antibody demonstrated that Zfp36l1/l2 nuclear localization was increased in the airway epithelium of an acute asthma mouse model, which was further enhanced in a chronic model. Immunostaining of human bronchial biopsies showed that airway epithelial cell staining of ZFP36L1 was decreased in severe asthma as compared with mild, while ZFP36L2 was upregulated. Restoring the levels of ZFP36L1 and ZFP36L2 in primary bronchial epithelial cells from patients with severe asthma decreased the mRNA expression of IL6, IL8 and CSF2.Discussion: We propose that the dysregulation of ZFP36L1/L2 levels as well as their subcellular mislocalization contributes to changes in mRNA expression and cytoplasmic fate in asthma.

Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b+ platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti–mouse CD49b-PE (phycoerythrin) antibody. Platelet–endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b+ platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism.

Chaperonin 60.1 (Cpn60.1) is a protein derived from Mycobacterium tuberculosis that has been shown, along with its peptide fragment IRL201104, to have beneficial effects in models of allergic inflammation. To further investigate the anti-inflammatory properties of Cpn60.1 and IRL201104, we have investigated these molecules in a model of nonallergic lung inflammation. Mice were treated with Cpn60.1 (0.5-5,000 ng/kg) or IRL201104 (0.00025-2.5 ng/kg), immediately before intranasal instillation of bacterial lipopolysaccharide (LPS). Cytokine levels and cell numbers in mouse bronchoalveolar lavage (BAL) fluid were measured 4 h after LPS administration. In some experiments, mice were depleted of lung-resident phagocytes. Cells from BAL fluid were analyzed for inflammasome function. Human umbilical vein endothelial cells (HUVECs) were analyzed for adhesion molecule expression. Human neutrophils were analyzed for integrin expression, chemotaxis, and cell polarization. Cpn60.1 and IRL201104 significantly inhibited neutrophil migration into the airways, independently of route of administration. This effect of the peptide was absent in TLR4 and annexin A1 knockout mice. Intravital microscopy revealed that IRL201104 reduced leukocyte adhesion and migration into inflamed tissues. However, IRL201104 did not significantly affect adhesion molecule expression in HUVECs or integrin expression, chemotaxis, or polarization of human neutrophils at the studied concentrations. In phagocyte-depleted animals, the anti-inflammatory effect of IRL201104 was not significant. IRL201104 significantly reduced IL-1 beta and NLRP3 expression and increased A20 expression in BAL cells. This study shows that Cpn60.1 and IRL201104 potently inhibit LPS-induced neutrophil infiltration in mouse lungs by a mechanism dependent on tissue-resident phagocytes and to a much lesser extent, the proresolving factor annexin A1.

BACKGROUND:We have previously demonstrated that Mycobacteria tuberculosis chaperonin 60.1 inhibits leucocyte diapedesis and bronchial hyperresponsiveness in a murine model of allergic lung inflammation.METHODS:In the present study, we have investigated the effect of a shorter peptide sequence derived from Cpn 60.1, named IRL201104, on allergic lung inflammation induced by ovalbumin (OVA) in mice and by house dust mite (HDM) in guinea pigs, as well as investigating the action of IRL201104 on human cells in vitro.RESULTS:Pre-treatment of mice or guinea pigs with IRL201104 inhibits the infiltration of eosinophils to the lung, cytokine release, and in guinea pig skin, inhibits allergen-induced vascular permeability. The protective effect of intranasal IRL201104 against OVA-induced eosinophilia persisted for up to 20 days post-treatment. Moreover, OVA-sensitized mice treated intranasally with 20 ng/kg of IRL201104 show a significant increase in the expression of the anti-inflammatory molecule ubiquitin A20 and significant inhibition of the activation of NF-κB in lung tissue. Our results also show that A20 expression was significantly reduced in blood leucocytes and ASM obtained from patients with asthma compared to cells obtained from healthy subjects which were restored after incubation with IRL201104 in vitro, when added alone, or in combination with LPS or TNF-α in ASM.CONCLUSIONS:Our results suggest that a peptide derived from mycobacterial Cpn60.1 has a long-lasting anti-inflammatory and immunomodulatory activity which may help explain some of the protective effects of TB against allergic diseases.

Purpose: The development of inhaled drug products is expensive and involves time-consuming pharmacokinetic (PK) and pharmacodynamic (PD) studies. There are few in vitro cell-based assays to evaluate the disposition and action of orally inhaled drugs to guide early product development and minimise risk. The aim of the present study was to develop a co-culture bioassay, combining an airway epithelial cell line (Calu-3) with cultured human primary airway smooth muscle cells (ASM), integrated with apparatus to deliver pharmaceutical aerosols. Methods: An assay for measuring cyclic adenosine monophosphate (cAMP) in ASM derived from healthy donors was adapted to provide a biochemical surrogate for ASM relaxation. Concentration-response curves for cAMP were established for three drugs that elicit ASM relaxation: isoprenaline (ISO), forskolin (FOR) and salbutamol sulphate. The ASM bioassay was incorporated into a co-culture model in which air-interfaced Calu-3 cell layers, representing the permeability barrier of the airway epithelium, were grown on transwell inserts above ASM cells cultured in the well of the base-plate. The sensitivity of this bioassay to salbutamol delivered using different formulations and aerosol products was evaluated. Results: ASM responded with concentration dependent increases in cAMP when exposed to 16(-9) to 10(-5) M ISO, FOR or salbutamol sulphate solutions for 15 or 30 min. Salbutamol formulated with different counter ions elicited differential cAMP responses in ASM (xinafoate > base = sulphate) suggesting that this bioassay could discriminate between formulations with different potency. A similar rank order of potency was observed for the different salbutamol salts when applied as aerosols to the co-culture model. Discussion: We have developed a novel bioassay using human ASM in co-culture with human respiratory epithelial cells to better mimic various elements that contribute to the rate and extent of local drug availability in the lungs following topical administration. The bioassay offers an opportunity to investigate the factors determining the activity of inhaled bronchodilator drugs in a more biologically relevant system than that has previously been described and with further development and validation, this novel bioassay could provide a method to guide the more efficient development of inhaled bronchodilators, reducing the current reliance on in vivo studies.

We report 5-substituted uridine derivatives as novel, uncharged inhibitors of β-1,4-galactosyltransferase and chemical tools for cellular applications. The new inhibitors reduce P-selectin glycoprotein 1 (PSGL-1) expression in human monocytes. Our results also provide novel insights into a unique mode of glycosyltransferase inhibition.

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a cell-surface glycoprotein that is expressed, either constitutively or inducibly, on all myeloid and lymphoid cell lineages. PSGL-1 is implicated in cell–cell interactions between platelets, leukocytes and endothelial cells, and a key mediator of inflammatory cell recruitment and transmigration into tissues. Here, we have investigated the effects of the β-1,4-galactosyltransferase inhibitor 5-(5-formylthien-2-yl) UDP-Gal (5-FT UDP-Gal, compound 1) and two close derivatives on the cell surface levels of PSGL-1 on human peripheral blood mononuclear cells (hPBMCs). PSGL-1 levels were studied both under basal conditions, and upon stimulation of hPBMCs with interleukin-1β (IL-1β). Between 1 and 24 hours after IL-1β stimulation, we observed initial PSGL-1 shedding, followed by an increase in PSGL-1 levels on the cell surface, with a maximal window between IL-1β-induced and basal levels after 72 h. All three inhibitors reduce PSGL-1 levels on IL-1β-stimulated cells in a concentration-dependent manner, but show no such effect in resting cells. Compound 1 also affects the cell surface levels of adhesion molecule CD11b in IL-1β-stimulated hPBMCs, but not of glycoproteins CD14 and CCR2. This activity profile may be linked to the inhibition of global Sialyl Lewis presentation on hPBMCs by compound 1, which we have also observed. Although this mechanistic explanation remains hypothetical at present, our results show, for the first time, that small molecules can discriminate between IL-1β-induced and basal levels of cell surface PSGL-1. These findings open new avenues for intervention with PSGL-1 presentation on the cell surface of primed hPBMCs and may have implications for anti-inflammatory drug development.

Although foamy macrophages (FMΦ) are commonly observed during nonclinical development of medicines for inhalation, there are no accepted criteria to differentiate adaptive from adverse FMΦ responses in drug safety studies. The purpose of this study was to develop a multiparameter in vitro assay strategy to differentiate and characterize different mechanisms of drug-induced FMΦ. Amiodarone, staurosporine, and poly(vinyl acetate) nanoparticles were used to induce distinct FMΦ phenotypes in J774A.1 cells, which were then compared with negative controls. Treated macrophages were evaluated for morphometry, lipid accumulation, gene expression, apoptosis, cell activation, and phagocytosis. Analysis of vacuolization (number/area vacuoles per cell) and phospholipid content revealed inducer-dependent distinctive patterns, which were confirmed by electron microscopy. In contrast to the other inducers, amiodarone increased vacuole size rather than number and resulted in phospholipid accumulation. No pronounced dysregulation of transcriptional activity or apoptosis was observed in response to sublethal concentrations of all inducers. Functionally, FMΦ induction did not affect macrophage activation by lipopolysaccharide, but it reduced phagocytic capacity, with different patterns of induction, severity, and resolution observed with the different inducers. An in vitro multiparameter assay strategy is reported that successfully differentiates and characterizes mechanisms leading to FMΦ induction by different types of agents.

Brain natriuretic peptide (BNP) relaxes airways by activating natriuretic peptide receptor-A and elevating cyclic guanosine monophosphate. BNP is more effective in passively sensitized human bronchi compared with control airways. The molecular and cellular patterns involved in this signaling are unknown. The aim of this study was to investigate the influence of BNP on airway smooth muscle (ASM) cells obtained from donors with asthma and healthy donors and to identify the mechanisms involved in BNP-mediated relaxation. The contractile response of ASM cells was microscopically assessed in vitro in the presence of 1 μM BNP or with supernatant from human bronchial epithelial (BEAS-2B) cells pretreated with 1 μM BNP. We investigated the role of muscarinic M2 receptors and inducible nitric oxide synthase (iNOS), quantified the release of acetylcholine and nitric oxide (NO), and assessed the gene/protein expression of iNOS and myosin phosphatase target subunit 1 (MYPT1). Supernatant from BEAS-2B cells treated with BNP reduced the hyperreactivity of asthmatic ASM cells by shifting the potency of histamine by 1.19-fold but had no effect in healthy ASM cells. BNP was not effective directly on ASM cells. Blocking muscarinic M2-receptors and iNOS abolished the protective role of supernatant from BEAS-2B treated with BNP. BNP stimulated the release of acetylcholine (210.7 ± 11.1%) from BEAS-2B cells that in turn increased MYPT1 and iNOS gene/protein expression and enhanced NO levels in asthmatic ASM supernatant (35.0 ± 13.0%). This study provides evidence that BNP protects against bronchial hyperresponsiveness via an interaction between respiratory epithelium and ASM in subjects with asthma.

In chronic lung disorders such as in asthma and chronic obstructive pulmonary disease (COPD) there is increased bronchial angiogenesis and remodelling of pulmonary vessels culminating to altered bronchial and pulmonary circulation. The involvement of residential cells such as endothelial cells, smooth muscle cells and pulmonary fibroblasts, all appear to have a crucial role in the progression of vascular inflammation and remodelling. The regulatory abnormalities, growth factors and mediators implicated in the pulmonary vascular changes of asthma and COPD subjects and potential therapeutic targets have been described in this review.

The biological responses of airway smooth muscle (ASM) are diverse, in part due to ASM phenotype plasticity. ASM phenotype plasticity refers to the ability of ASM cells to change the degree of a variety of functions, including contractility, proliferation, migration and secretion of inflammatory mediators. This plasticity occurs due to intrinsic or acquired abnormalities in ASM cells, and these abnormalities or predisposition of the ASM cell may alter the ASM response and in some cases recapitulate disease hallmarks of asthma. These phenotypic changes are ultimately determined by multiple stimuli and occur due to alterations in the intricate balance or reversible state that maintains ASM cells in either a contractile or synthetic state, through processes termed maturation or modulation, respectively. To elucidate the role of ASM phenotype in disease states, numerous in vitro studies have suggested a phenotypic switch in ASM primary cell cultures as an explanation for the plethora of responses mediated by ASM cells. Moreover, there is overwhelming evidence suggesting that the immunomodulatory response of ASM is due to the acquisition of a synthetic phenotype; however, whether this degree of plasticity is present in vivo as opposed to cell culture-based models remains speculative. Nonetheless, this review will give an overall scope of ASM phenotypic markers, triggers of ASM phenotype modulation and novel therapeutic approaches to control ASM phenotype plasticity.

Introduction Airway smooth muscle cells (ASM) from subjects with mild asthma secrete increased levels of VEGF and increased expression of adenosine A1 receptor in bronchial biopsies compared to healthy subjects. We hypothesised that VEGF release from ASM in asthma is mediated via adenosine A1 receptors. Objectives To examine the effect of adenosine on VEGF release in ASM derived from asthmatic subjects. Methods ASM from healthy (n=4) and mild asthmatic (n=3) subjects were treated with and without adenosine receptor agonists (0.01-100µM) targeting A1 (CPA), A2 (CV1808), A2a (CGS21680), A3 (IB-MECA) or non-selective (adenosine, NECA). ASM were also treated with 10µM NECA following pre-treatment with adenosine receptor antagonists (1-100nM). After 24hr, VEGF levels in cell-conditioned media were determined by ELISA. Results Constitutive levels of VEGF in ASM derived from asthmatics (275±34pg/ml/10 6 cells) were 1.7 fold greater than ASM derived from healthy subjects (159±34pg/ml/10 6 cells, p Conclusions VEGF release induced by adenosine receptor activation in ASM derived from mild asthmatic subjects was mediated through A2a and A2b receptors.

Smooth muscle comprises a key functional component of both the airways and their supporting vasculature. Dysfunction of smooth muscle contributes to and exacerbates a host of breathing-associated pathologies such as asthma, chronic obstructive pulmonary disease and pulmonary hypertension. These diseases may be marked by airway and/or vascular smooth muscle hypertrophy, proliferation and hyper-reactivity, and related conditions such as fibrosis and extracellular matrix remodeling. This review will focus on the contribution of airway or vascular smooth dysfunction to common airway diseases.

In asthma, the airway smooth muscle (ASM) cell plays a central role in disease pathogenesis through cellular changes which may impact on its microenvironment and alter ASM response and function. The answer to the long debated question of what makes a 'healthy' ASM cell become 'asthmatic' still remains speculative. What is known of an 'asthmatic' ASM cell, is its ability to contribute to the hallmarks of asthma such as bronchoconstriction (contractile phenotype), inflammation (synthetic phenotype) and ASM hyperplasia (proliferative phenotype). The phenotype of healthy or diseased ASM cells or tissue for the most part is determined by expression of key phenotypic markers. ASM is commonly accepted to have different phenotypes: the contractile (differentiated) state versus the synthetic (dedifferentiated) state (with the capacity to synthesize mediators, proliferate and migrate). There is now accumulating evidence that the synthetic functions of ASM in culture derived from asthmatic and non-asthmatic donors differ. Some of these differences include an altered profile and increased production of extracellular matrix proteins, pro-inflammatory mediators and adhesion receptors, collectively suggesting that ASM cells from asthmatic subjects have the capacity to alter their environment, actively participate in repair processes and functionally respond to changes in their microenvironment.

Glucose moves into airway secretions after a glucose load. Therefore people with diabetes or hyperglycemia spend a significant proportion of each day with glucose in their airways secretions. This study investigated the effects of glucose on isolated human airways and on cultured airway smooth muscle (ASM) cells. Human isolated bronchi were stimulated with acetylcholine, histamine, and transmural stimulation and treated with the selective ROCK inhibitors Y27632 and SB772077B under high-glucose conditions. The effect of high glucose concentrations on intracellular calcium flux and the phosphorylation of MYPT1 in ASM cells was also investigated. High (44 mM for 6 h) glucose, but not mannitol, concentrations led to an enhanced responsiveness of ASM to contractile agents. Y27632 and SB772077B completely abolished (P < 0.05) the enhanced contractile effects with a high-concentration glucose solution, compared with control tissues. In cultured ASM cells, incubation with high glucose concentrations significantly (P < 0.05) enhanced bradykinin-induced intracellular calcium flux and the levels of pMYPT1, which were inhibited by Y27632 (P < 0.05). Our study has demonstrated that high glucose concentrations leads to hyperresponsiveness of human isolated bronchi and enhances intracellular calcium release in cultured ASM cells via a Rho/ROCK- and pMYPT1-dependent pathway, suggesting that this crucial pathway may contribute to the reduced lung function observed in patients with diabetes. These data propose novel targets for the treatment of patients with respiratory diseases that also suffer from diabetes mellitus.

RATIONALE: Asthmatic airways have an increased number and size of vascular structures, which contribute to airflow obstruction and hyperresponsiveness. We have examined if pro-angiogenic mediators are present in BALf from asthmatics.METHODS: 11 steroid naïve asthmatic, and 13 healthy volunteers were recruited. Angiogenic activity of BALf was evaluated using an in vitro angiogenesis assay (AngioKit; TCS Cell Works) comprising a co-culture of human endothelial cells with dermal fibroblasts. BALf was fractionated through a Centricon Concentrator with a 10kD exclusion limit. Aliquots of BALf were added to culture wells on days 1, 4, 7, and 9. Cell monolayers were fixed on day 11 and vascular structures were visualised by labelling with anti-CD31. Vascular formation was quantified using computerized image analysis (AngioSys;TCS).RESULTS: Healthy BALf induced the formation of CD31 positive vascular structures that was 3-fold greater than with culture medium alone. The angiogenic activity induced by asthmatic BALf was 2 to 3-fold greater than healthy BALf. Vascular branching points were increased 3-fold with BALf from asthmatics compared to healthy controls (p<0.01). The number of vascular tubules was also greater in asthmatic culture (p<0.05) as was the total length of vascular tubules (p<0.001).CONCLUSIONS: Pro-angiogenic activity is present in BALf from healthy subjects and is markedly elevated in patients with asthma. Identifying the key angiogenic factors present, their source and regulation may result in future therapeutic strategies for treating asthma. RATIONALE: Asthmatic airways have an increased number and size of vascular structures, which contribute to airflow obstruction and hyperresponsiveness. We have examined if pro-angiogenic mediators are present in BALf from asthmatics. METHODS: 11 steroid naïve asthmatic, and 13 healthy volunteers were recruited. Angiogenic activity of BALf was evaluated using an in vitro angiogenesis assay (AngioKit; TCS Cell Works) comprising a co-culture of human endothelial cells with dermal fibroblasts. BALf was fractionated through a Centricon Concentrator with a 10kD exclusion limit. Aliquots of BALf were added to culture wells on days 1, 4, 7, and 9. Cell monolayers were fixed on day 11 and vascular structures were visualised by labelling with anti-CD31. Vascular formation was quantified using computerized image analysis (AngioSys;TCS). RESULTS: Healthy BALf induced the formation of CD31 positive vascular structures that was 3-fold greater than with culture medium alone. The angiogenic activity induced by asthmatic BALf was 2 to 3-fold greater than healthy BALf. Vascular branching points were increased 3-fold with BALf from asthmatics compared to healthy controls (p<0.01). The number of vascular tubules was also greater in asthmatic culture (p<0.05) as was the total length of vascular tubules (p<0.001). CONCLUSIONS: Pro-angiogenic activity is present in BALf from healthy subjects and is markedly elevated in patients with asthma. Identifying the key angiogenic factors present, their source and regulation may result in future therapeutic strategies for treating asthma.