Vitamin D deficiency is common in patients with chronic obstructive pulmonary disease (COPD), yet a comprehensive analysis of environmental and genetic determinants of serum 25-hydroxyvitamin D (25 [OW) concentration in patients with this condition is lacking. We conducted a multi-centre cross-sectional study in 278 COPD patients aged 41-92 years in London, UK. Details of potential environmental determinants of vitamin D status and COPD symptom control and severity were collected by questionnaire, and blood samples were taken for analysis of serum 25(OH)D concentration and DNA extraction. All participants performed spirometry and underwent measurement of weight and height. Quadriceps muscle strength (QS) was measured in 134 participants, and sputum induction with enumeration of lower airway eosinophil and neutrophil counts was performed for 44 participants. Thirty-seven single nucleotide polymorphisms (SNP) in 11 genes in the vitamin D pathway (DBP, DHCR7, CYP2R1, CYP27B1, CYP24A1, CYP27A1, CYP3A4, LRP2, CUBN, RXRA, and VDR) were typed using Taqman allelic discrimination assays. Linear regression was used to identify environmental and genetic factors independently associated with serum 25(OH)D concentration and to determine whether vitamin D status or genetic factors independently associated with % predicted forced expiratory volume in one second (FEV1), % predicted forced vital capacity (FVC), the ratio of FEV1 to FVC (FEV1:FVC), daily inhaled corticosteroid (ICS) dose, respiratory quality of life (QoL), QS, and the percentage of eosinophils and neutrophils in induced sputum. Mean serum 25(OH)D concentration was 45.4 nmol/L (SD 25.3); 171/278 (61.5%) participants were vitamin D deficient (serum 25[OHID concentration <50 nmol/L). Lower vitamin D status was independently associated with higher body mass index (P=0.001), lower socio-economic position (P=0.037), lack of vitamin D supplement consumption (P<0.001), sampling in Winter or Spring (P for trend = 0.006) and lack of a recent sunny holiday (P=0.002). Vitamin D deficiency associated with reduced % predicted FEV1 (P for trend = 0.060) and % predicted FVC (P for trend = 0.003), but it did not associate with FEV1:FVC, ICS dose, QoL, QS, or the percentage of eosinophils or neutrophils in induced sputum. After correction for multiple comparisons testing, genetic variation in the vitamin D pathway was not found to associate with serum 25(OH)D concentration or clinical correlates of COPD severity. Vitamin D deficiency was common in this group of COPD patients in the UK, and it associated independently with reduced % predicted FEV1 and FVC. However, genetic variation in the vitamin D pathway was not associated with vitamin D status or severity of COPD. (C) 2017 Elsevier Ltd. All rights reserved.

Vitamin D deficiency is common in children with asthma, and it associates with poor asthma control, reduced forced expiratory volume in one second (FEV1) and increased requirement for inhaled corticosteroids (ICS). Cross-sectional studies investigating the prevalence, determinants and clinical correlates of vitamin D deficiency in adults with asthma are lacking. We conducted a multi-centre cross-sectional study in 297 adults with a medical record diagnosis of ICS-treated asthma living in London, UK. Details of potential environmental determinants of vitamin D status, asthma control and medication use were collected by questionnaire; blood samples were taken for analysis of serum 25(OH)D concentration and DNA extraction, and participants underwent measurement of weight, height and fractional exhaled nitric oxide concentration (FeNO), spirometry and sputum induction for determination of lower airway eosinophil counts (n=35 sub-group). Thirty-five single nucleotide polymorphisms (SNP) in 11 vitamin D pathway genes (DBP, DHCR7, RXRA, CYP2R1, CYP27B1, CYP24A1, CYP3A4 CYP27A1, LRP2, CUBN, VDR) were typed using Taqman allelic discrimination assays. Linear regression was used to identify environmental and genetic factors independently associated with serum 25(OH)D concentration, and to determine whether vitamin D status was independently associated with Asthma Control Test (ACT) score, ICS dose, FeNO, forced vital capacity (FVC), FEV1 or lower airway eosinophilia. Mean serum 25(OH)D concentration was 50.6nmol/L (SD 24.9); 162/297 (54.5%) participants were vitamin D deficient (serum 25(OH)D concentration <50nmol/L). Lower vitamin D status was associated with higher body mass index (P=0.014), non-White ethnicity (P=0.036), unemployment (P for trend=0.012), lack of vitamin D supplement use (P<0.001), sampling in Winter or Spring (P for trend <0.001) and lack of a recent sunny holiday abroad (P=0.030), but not with potential genetic determinants. Vitamin D status was not found to associate with any marker of asthma control investigated. Vitamin D deficiency is common among UK adults with ICS-treated asthma, and classical environmental determinants of serum 25(OH)D operate in this population. However, in contrast to studies conducted in children, we found no association between vitamin D status and markers of asthma severity or control.

Rationale Asthma exacerbations are commonly precipitated by viral upper respiratory infections (URIs). Vitamin D insufficiency associates with susceptibility to URI in patients with asthma. Trials of vitamin D in adults with asthma with incidence of exacerbation and URI as primary outcome are lacking. Objective To conduct a randomised controlled trial of vitamin D3 supplementation for the prevention of asthma exacerbation and URI (coprimary outcomes). Measurements and methods 250 adults with asthma in London, UK were allocated to receive six 2-monthly oral doses of 3 mg vitamin D3 (n=125) or placebo (n=125) over 1 year. Secondary outcomes included asthma control test and St George's Respiratory Questionnaire scores, fractional exhaled nitric oxide and concentrations of inflammatory markers in induced sputum. Subgroup analyses were performed to determine whether effects of supplementation were modified by baseline vitamin D status or genotype for 34 single nucleotide polymorphisms in 11 vitamin D pathway genes. Main results 206/250 participants (82%) were vitamin D insufficient at baseline. Vitamin D3 did not influence time to first severe exacerbation (adjusted HR 1.02, 95% CI 0.69 to 1.53, p=0.91) or first URI (adjusted HR 0.87, 95% CI 0.64 to 1.16, p=0.34). No clinically important effect of vitamin D3 was seen on any of the secondary outcomes listed above. The influence of vitamin D3 on coprimary outcomes was not modified by baseline vitamin D status or genotype. Conclusions Bolus-dose vitamin D3 supplementation did not influence time to exacerbation or URI in a population of adults with asthma with a high prevalence of baseline vitamin D insufficiency. Trial registration number NCT00978315 (ClinicalTrials.gov).

Background: New treatments for asthma & COPD may require evaluation in sub-groups with lower airway eosinophilia or neutrophilia. A non-invasive test to identify these phenotypes may reduce unnecessary sputum induction at screening. Aim: To establish whether eosinophil and neutrophil counts correlate with those in the lower airway in asthma and COPD patients. Methods: 35 patients with ICS-treated asthma and 44 patients with COPD underwent sputum induction and phlebotomy on ≤ 3 occasions over 1 yr. Differential white cell counts were performed on peripheral blood and induced sputum; Spearman9s test was used to assess a correlation. Results: 97 paired samples from 35 asthma patients; 111 paired samples from 44 COPD patients were analysed. Proportion of eosinophils in peripheral blood correlated positively with proportion of eosinophils in induced sputum in asthma (r =0.61, 95% CI 0.47 to 0.73, P<0.0001) and COPD (r =0.46, 95% CI 0.29 to 0.60, P<0.0001). Sensitivity of peripheral blood eosinophilia ≥4% for identification of lower airway eosinophilia >3% was 74% in asthma and 42% in COPD; specificity was 84% in both asthma and COPD; positive predictive value was 78% in asthma and 56% in COPD; and negative predictive value was 81% in asthma and 75% in COPD. There was no correlation between proportion of neutrophils in peripheral blood vs. induced sputum (for asthma, r =-0.18, 95% CI 0.37 to 0.03, P=0.08; for COPD, r=0.15, 95% CI -0.05 to 0.33, P=0.13). Conclusions: Eosinophil counts in induced sputum were positively correlated with those in peripheral blood in patients with asthma and COPD. A peripheral blood eosinophilia screening test could be used to identify patients with lower airway eosinophilia.

Background Patients with chronic obstructive pulmonary disease (COPD) often have vitamin D deficiency, which is associated with increased susceptibility to upper respiratory infection-a major precipitant of exacerbation. Multicentre trials of vitamin D supplementation for prevention of exacerbation and upper respiratory infection in patients with COPD are lacking. We therefore investigated whether vitamin D-3 (colecakiferol) supplementation would reduce the incidence of moderate or severe COPD exacerbations and upper respiratory infections.Methods We did a randomised, double-blind, placebo-controlled trial of vitamin D, supplementation in adults with COPD in 60 general practices and four Acute National Health Service Trust clinics in London, UK. Patients were allocated to receive six 2-monthly oral doses of 3 mg vitamin D-3 or placebo over 1 year in a 1:1 ratio using computer-generated permuted block randomisation. Participants and study staff were masked to treatment assignment. Coprimary outcomes were time to first moderate or severe exacerbation and first upper respiratory infection. Analysis was by intention to treat. A prespecified subgroup analysis was done to assess whether effects of the intervention on the coprimary outcomes were modified by baseline vitamin D status. This trial is registered with ClinicalTrials.gov, number NCT00977873.Findings 240 patients were randomly allocated to the vitamin D-3 group (n=122) and placebo group (n=118). Vitamin D, compared with placebo did not affect time to first moderate or severe exacerbation ( adjusted hazard ratio 0.86, 95% CI 0.60-1.24, p=0.42) or time to first upper respiratory infection (0.95, 0.69-1.31, p=0.75). Prespecified subgroup analysis showed that vitamin D-3 was protective against moderate or severe exacerbation in participants with baseline serum 25-hydroxyvitamin D concentrations of less than 50 nmol/L (0.57, 0.35-0.92, p=0.021), but not in those with baseline 25-hydroxyvitamin D levels of at least 50 nmol/L (1.45, 0.81-2.62, p=0.21; p=0.021 for interaction between allocation and baseline serum 25-hydroxyvitamin D status). Baseline vitamin D status did not modify the effect of the intervention on risk of upper respiratory infection (p(interaction)=0.41).Interpretation Vitamin D-3 supplementation protected against moderate or severe exacerbation, but not upper respiratory infection, in patients with COPD with baseline 25-hydroxyvitamin D levels of less than 50 nmol/L. Our findings suggest that correction of vitamin D deficiency in patients with COPD reduces the risk of moderate or severe exacerbation.

Introduction and objectives Inadequate vitamin D status is common in patients with COPD, and it associates with susceptibility to upper respiratory infection (URI) – a major precipitant of exacerbation. Multi-centre trials of vitamin D supplementation for prevention of exacerbation and URI in COPD are lacking. We therefore conducted a multi-centre double-blind randomised placebo-controlled trial of vitamin D supplementation for the prevention of moderate/severe exacerbation and URI in adults with COPD. Methods Two hundred and forty patients were allocated to receive a 2-monthly oral dose of 3 mg vitamin D3 or placebo for one year. Co-primary outcomes were time to first moderate/severe exacerbation and time to first URI. Secondary outcomes included peak severity and area under the curve for exacerbation symptoms. A pre-specified sub-group analysis was conducted to determine whether effects of the intervention on co-primary outcomes were modified by baseline vitamin D status. This trial is registered with ClinicalTrials.gov (NCT00977873). Results 122 participants were allocated to the intervention arm of the trial, and 118 to the control arm. Vitamin D supplementation did not influence time to first exacerbation (HR 0.86, 95% CI 0.60–1.24, p = 0.42) or time to first URI (HR 0.95, 95% CI 0.69–1.31, p = 0.75) in the study population as a whole, but it did reduce peak severity (p = 0.042) and area under the curve (p = 0.032) for exacerbation symptoms. Pre-specified sub-group analysis revealed that vitamin D supplementation was protective against moderate/severe exacerbation among the 148 participants with baseline serum 25-hydroxyvitamin D (25[OH]D) concentration < 50 nmol/L (aHR 0.57, 95% CI 0.35 to 0.92, p = 0.021), but not among the 92 participants with baseline serum 25(OH)D ≥ 50 nmol/L (aHR 1.45, 95% CI 0.81 to 2.62, p = 0.21; P for interaction = 0.021). Baseline vitamin D status did not modify the effect of vitamin D supplementation on risk of URI (P for interaction = 0.41). Conclusions Vitamin D supplementation protected against moderate/severe exacerbation, but not upper respiratory infection, in COPD patients with baseline 25(OH)D < 50 nmol/L. It also modestly reduced peak severity and area under the curve for exacerbation symptom scores, irrespective of baseline vitamin D status.

Introduction and objectives Asthma exacerbations are commonly precipitated by viral upper respiratory infections (URI). Vitamin D insufficiency associates with susceptibility to URI in patients with asthma. A recent vitamin D trial in adults with asthma reported a trend towards reduced exacerbation risk in the intervention arm as a secondary outcome. Trials of vitamin D in adults with asthma with incidence of exacerbation and URI as primary outcome are lacking. We therefore conducted a multi-centre randomised controlled trial of vitamin D3 supplementation in adults with asthma with co-primary outcomes of severe exacerbation and URI. Methods Two hundred and fifty adults with inhaled corticosteroid (ICS)-treated asthma were allocated to receive six 2-monthly oral doses of 3 mg vitamin D3 or placebo over one year. Co-primary outcomes were time to first severe exacerbation and time to first URI. Sub-group analyses were performed to determine whether effects of supplementation were modified by baseline vitamin D status or genotype for thirty-four single nucleotide polymorphisms in eleven vitamin D pathway genes. Results Participants were allocated to vitamin D3 vs. placebo in equal numbers; 82% were vitamin D insufficient at baseline. Vitamin D3 supplementation did not influence time to first severe exacerbation (aHR 1.02, 95% CI 0.69–1.53, P = 0.91) or time to first URI (aHR 0.87, 95% CI 0.64–1.16, P = 0.34). The influence of vitamin D3 on co-primary outcomes was not modified by baseline vitamin D status or genotype. Of 16 pre-specified secondary outcomes, only one showed a difference between arms: vitamin D supplementation induced a modest improvement in respiratory quality of life as evidenced by a reduction in mean total score for the St George’s Respiratory Questionnaire at 2 months (-3.9 points, p = 0.005), 6 months (-3.7 points, p = 0.038) and 12 months (-3.3 points, p = 0.060). Conclusions Vitamin D3 supplementation did not influence time to exacerbation or URI in a population of adults with ICS-treated asthma with a high prevalence of baseline vitamin D insufficiency.

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.

A 22-year-old woman was admitted unwell with headache, fever and encephalopathy. She was born in the UK to parents from Hong Kong. She had a past history of asthma, perennial rhinitis and atopic dermatitis for which she took a budesonide 100 μg turbohaler and beclomethasone nasal spray. Three weeks before admission she had returned from holiday in Valencia and shortly after her return developed a 'flu-like illness followed by worsening asthma symptoms. Despite increasing usage of her corticosteroid turbohaler to eight puffs a day, her cough and wheeze worsened. Her GP prescribed a 5-day course of prednisolone 50 mg daily (approximately 1 mg/kg). Her asthma symptoms improved but she developed a headache 2 days after finishing the corticosteroid course. Over the next 5 days she became more unwell with worsening headache …

Phenotypic modulation of airway smooth muscle (ASM) is an important feature of airway remodeling in asthma that is characterized by enhanced proliferation and secretion of pro-inflammatory chemokines. These activities are regulated by the concentration of free Ca(2+) in the cytosol ([Ca(2+)](i)). A rise in [Ca(2+)](i) is normalized by rapid reuptake of Ca(2+) into sarcoplasmic reticulum (SR) stores by the sarco/endoplasmic reticulum Ca(2+) (SERCA) pump. We examined whether increased proliferative and secretory responses of ASM from asthmatics result from reduced SERCA expression. ASM cells were cultured from subjects with and without asthma. SERCA expression was evaluated by western blot, immunohistochemistry and real-time PCR. Changes in [Ca(2+)](i), cell spreading, cellular proliferation, and eotaxin-1 release were measured. Compared with control cells from healthy subjects, SERCA2 mRNA and protein expression was reduced in ASM cells from subjects with moderately severe asthma. SERCA2 expression was similarly reduced in ASM in vivo in subjects with moderate/severe asthma. Rises in [Ca(2+)](i) following cell surface receptor-induced SR activation, or inhibition of SERCA-mediated Ca(2+) re-uptake, were attenuated in ASM cells from asthmatics. Likewise, the return to baseline of [Ca](i) after stimulation by bradykinin was delayed by approximately 50% in ASM cells from asthmatics. siRNA-mediated knockdown of SERCA2 in ASM from healthy subjects increased cell spreading, eotaxin-1 release and proliferation. Our findings implicate a deficiency in SERCA2 in ASM in asthma that contributes to its secretory and hyperproliferative phenotype in asthma, and which may play a key role in mechanisms of airway remodeling.

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.

RATIONALE:Airway remodeling in asthma involves accumulation of airway smooth muscle (ASM) and increased vascularity due to angiogenesis. Bronchial blood vessels and ASM are found in close proximity, and ASM releases multiple proinflammatory mediators, including vascular endothelial growth factor (VEGF).OBJECTIVES:We examined whether release of proangiogenic mediators is increased in ASM from subjects with asthma and whether this is translated to induction of angiogenesis.METHODS:Biopsy-derived ASM cells were cultured from 12 subjects with mild asthma, 8 with moderate asthma, and 9 healthy control subjects. Angiogenesis induced by cell-conditioned medium (CM) from ASM was evaluated in a tubule formation assay. Anti-CD31-labeled tubules were quantified by image analysis. Angiogenic factors in CM were quantified by antibody arrays and by enzyme-linked immunosorbent assay.MEASUREMENTS AND MAIN RESULTS:Induction of angiogenesis by CM from unstimulated ASM was increased in subjects with mild asthma (twofold) and moderate asthma (threefold), compared with healthy CM (P < 0.001). Levels of angiogenic factors (VEGF, angiopoietin [Ang]-1, angiogenin) were similarly elevated in CM from subjects with asthma compared with that from healthy subjects (P < 0.05), whereas antiangiogenic factors (endostatin, Ang-2) were unchanged. VEGF, Ang-1, and angiogenin in combination increased vascularity (twofold, P < 0.01) in cultured intact biopsies. Selective VEGF immunodepletion abolished enhanced tubule formation by CM from asthmatic ASM (P < 0.01), but CM depletion of Ang-1 or angiogenin had no effect.CONCLUSIONS:ASM cultured from subjects with mild or moderate asthma, but not from healthy control subjects, promotes angiogenesis in vitro. This proangiogenic capacity resides in elevated VEGF release and suggests that ASM regulates airway neovascularization in asthma.

Rationale: Asthmatic airways have an increased number and size of vascular structures, which contribute to airflow obstruction and hyperresponsiveness.Objectives: We examined whether proangiogenic mediators are elevated in bronchoalveolar lavage fluid (BALF) from subjects with asthma and if this translated to induction of angiogenesis.Methods: Angiogenic activity in BALF from 12 healthy, nonatopic subjects and 10 atopic subjects with mild asthma was evaluated by examining tubule formation at 11 days in cocultures of human endothelial cells with dermal fibroblasts. Vascular structures were visualized by anti-CD31 labeling and quantified by image analysis. Angiogenic growth factors in BALF from healthy subjects and subjects with asthma were identified using antibody arrays and by ELISA.Measurements and Main Results: Angiogenic activity induced by BALF from healthy subjects was not different from basal tubule formation (p > 0.05). However, induction of tubular structures by asthmatic BALF was 2.5-fold greater (p < 0.001) compared with healthy samples. Similarly, levels of proangiogenic growth factors (angiogenin, vascular endothelial growth factor [VEGF], monocyte chemotactic protein-1) were increased approximately 2.5-fold (p < 0.05) in BALF from subjects with asthma, whereas antiangiogenic factors (endostatin, Ang-2) were unchanged. A blocking anti-VEGF antibody abolished tubule formation induced by BALF from either healthy subjects or subjects with asthma (p < 0.01). Immunodepletion of VEGF had no effect on basal tubule formation induced by healthy BALF but abrogated enhanced tubule formation by asthmatic BALF (p < 0.01).Conclusions: BALF collected from subjects with asthma but not healthy subjects is functionally active in promoting angiogenesis in vitro. The proangiogenic capacity of BALF from subjects with asthma resides in elevated VEGF derived from asthmatic airways. This observation supports VEGF as a key factor in vascular remodeling in asthma.