Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 86-92). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Lung cancer is the leading cause of cancer related death worldwide; despite recent treatment developments survival rates remain poor and are closely related to the patient’s clinical stage. Even among patients with early-stage lung cancer, which is amenable to surgical resection, prognosis is highly variable; some go on to live disease-free for many years whereas others quickly recur. Although post-operative chemotherapy is available it has associated morbidities and it is unclear which patients would benefit; therefore, there is a need for more effective stratification of patients. The adenocarcinoma sub-type of lung cancer is known to be morphologically heterogeneous however the majority of observed growth patterns, assessed by light microscopy, can be characterised into one of five formations: lepidic, papillary, acinar, solid and micropapillary. The morphology of each tumour has been proposed as a marker of prognosis and several studies have published a link between the most prevalent growth pattern and prognosis; suggesting those with predominantly solid or micropapillary tumours to have the least favourable outcomes. Indeed, it is now recommended that the proportion of each growth pattern and the predominant growth pattern should be reported for all resected lung adenocarcinomas; although no differential treatments have been recommended based on this assessment. The aim of this study was to determine whether combining the analysis of clinicopathological; morphological; and candidate protein, molecular genetic and transcriptomic characteristics in a single cohort of 208 early-stage, resected, adenocarcinomas with clinical follow-up could be used to identify a subset of patients at high risk of recurrence. Comprehensive morphological analysis was carried out including the presence, proportion and number of individual growth patterns; the predominant growth pattern as well as features previously associated with tumour grade (the presence of large numbers of mitotic figures, apoptotic bodies, inflammatory cells, prominent nucleoli, pleomorphic tumour cells, dyscohesive tumour cells and large amounts of necrosis and scar tissue within the tumour). In addition, gene expression was assessed using a panel of 31 cell-cycle related genes, EGFR and KRAS mutation status was determined, and EGFR and TTF1 protein expression investigated. In this study the predominant growth pattern defined by histopathology showed no ability to identify a group of patients with a poorer prognosis either in univariable or multivariable analysis. Univariable analysis identified nodal status [hazard ratio of N1 compared to N0 was 2.16 (95% CI 1.48 to 3.16, p< 0.0005)], clinical stage [hazard ratios of stage IIa and IIb compared to stage Ia were 3.15 (95% CI 1.73 to 5.73, p< 0.0005) and 2.22 (95% CI 1.10 to 4.48, p= 0.025) respectively], the presence of a significant amount of the papillary growth pattern [the hazard ratio of those with less than 8.5% papillary pattern was 0.657 (95% CI 0.44 to 0.98, p= 0.035)], and overall tumour grade score (including an assessment of necrosis, mitosis, apoptosis, nucleoli, scar tissue and inflammatory cells) [hazard ratio 1.71 (95% CI 1.14 to 2.56, p= 0.008)] as significantly associated with prognosis. Multivariable analysis using Cox’s proportional hazards model identified clinical stage (p< 0.0005), the presence of a significant amount of the papillary growth pattern (p= 0.048) and the presence of large numbers of mitotic figures (p=0.029) and apoptotic bodies (p= 0.015) as independently associated with disease specific survival; although after correction for type I errors only clinical stage remained significantly associated with prognosis with patients with stage Ia disease having a significantly better outcomes [hazard ratio 0.418 (95% CI 0.20 to 0.86)]. Classification and regression tree analysis (CART) was used to further explore the data and to develop decision trees for the prognostication of early-stage lung adenocarcinoma patients. Receiver operating characteristic analysis based on 5- year survival showed a minimal improvement in the area under the curve between a model utilizing currently available clinicopathologic characteristics only [nodal status and lesion size, (area under the curve 0.704, 95% CI 0.631 to 0.777)] and one including growth pattern characteristics [area under the curve 0.725, 95% CI 0.654 to 0.796]. The greatest improvement in prognostic accuracy was observed when gene expression analysis was included in the analysis [area under the curve 0.749, 95% CI 0.673 to 0.825]; however even this showed very little impact compared to routinely used clinicopathologic variables. This analysis suggests that the recommended characterisation of lung adenocarcinoma histology is not a robust predictor of patient outcomes; even a broader model which also included indicators of tumour grade and molecular characteristics was unable to identify a model sufficiently robust to implement into clinical practice and thereby potentially alter patient treatment. Currently routinely collected clinical characteristics; including nodal status, size and clinical stage; continue to provide the most robust method of prognostication and detailed and time-consuming morphological analysis offers no significant benefit to the patient.

Mammalian homolog Tribbles (Trbs) is a newly characterized protein family that includes three different isoforms: Trb1, Trb2, and Trb3. Tribbles are serine/threonine kinases lacking catalytic activity, thus their classification as pseudokinases. Despite their catalytic inactivity, Tribbles can interact with different proteins and regulate different biological functions. The most studied tribble family member, Trb3, was reported to play a major role in Drosophila's ventral furrow formation. Further studies revealed that Trb3 is also involved in diabetes, stress-response, and development. Previously, Trb3 upregulation was detected in certain types of cancer but its function remains unknown. The goal of our study is to gain a better understanding of the biological function of Trb3 in cancer, including the molecular mechanism of action. Using the cohort analysis, we identified higher levels of Trb3 in the lung tumor compared to the normal tissue. Furthermore, higher Trb3 expression in the lung tissue was associated with a poor survival in cancer patients. Silencing of Trb3 in A549 promoted cell growth. On the other hand, overexpression of Trb3 in NCI-H358 inhibited cell growth. The analysis of cell cycle gene profiling revealed a decrease in several genes that are essential for cell cycle progression in S phase in Trb3 overexpressed NCI-H358. The cell proliferation protein, Ki67, was also decreased in Trb3 overexpressed NCI-H358 cells. Moreover, Tb3 overexpressed cells formed higher colony number in soft agar assay and depicted higher migration ability in the Boyden chamber assay. Mesenchymal markers SNAIL, TWIST and N-cadherin were upregulated while epithelial E-cadherin was significantly reduced. Interestingly, prosurvival protein Akt was also reduced post Trb3 overexpression. Trb3 expression was associated with a poor survival. However, we discovered that Trb3 overexpression inhibited cell growth. Thus, we hypothesized that Trb3 expression might contribute to tumorigenesis during cellular metabolic stress. In order to understand the potential role of Trb3 in metabolic stress, NCI-H358 cells were treated with five different cellular stressors to mimic the tumor microenvironment. All stressors used were shown to induce endogenous Trb3 expression. Moreover, stress proteins ATF4, CHOP and ASNS were induced by all stressors. One of the stressors used was rotenone, an inhibitor of the complex I of the electron transport chain. Rotenone treatment induced Trb3 expression. This expression inversely correlated with cytochrome C expression. Furthermore, Trb3 expression positively correlated with the expression of mitophagic genes PINK1, Parkin and p62, which suggest that Trb3 is induced during ROS-mediated oxidative stress to participate in the clearing of damaged mitochondria. This targeted clearing of the mitochondria, a process known as mitophagy is essential for the cell survival of the lung cancer cells. Last, Trb3 overexpression rendered cancer cells resistant to docetaxel and cisplatin, two chemotherapeutic drugs used in lung cancer treatment. On the other hand, Trb3 depleted cells were more sensitive to the drugs. Our results suggest that Trb3 is activated in the primary tumor to promote metabolic adaptation through cell cycle arrest and the inhibition of aerobic glucose metabolism through Akt inhibition. Furthermore, Trb3 is essential during cell survival post ROS-mediated stress and participates in the clearing of damaged mitochondria during mitophagy. Last, stress-mediated activation of Trb3 confers lung cancer cells with chemoresistance and suggest that Trb3 could be a potential target in lung cancer therapy.

Rationale: Chronic obstructive pulmonary disease (COPD) is one of the leading causes of deaths worldwide. Patients with COPD have episodes of aggravated symptoms called exacerbations caused by pathogens or pollution. Respiratory viruses are associated with a significant number of COPD exacerbations with the most common virus being the rhinovirus (RV). The mechanisms by which RVs trigger COPD exacerbations are still unclear. Using human whole lung tissue explants (WTE), a novel model of RV-induced COPD exacerbations is proposed. Methods: WTE from COPD patients and smokers were initially stimulated with TLR ligands that are known to activate the same receptors as RV: poly(I:C) for TLR3 and R848 for TLR7/8 activation. Pro-inflammatory cytokines and type I and III IFN gene expression was measured by ELISA and qRT-PCR, respectively. A neutralising antibody against TNFα, a corticosteroid, and a panel of inhibitors targeting TLR pathway (p38 MAPK, IKK-2 and IRAK1/4) was applied to the tissue from COPD patients to establish which signalling pathways are responsible for the inflammatory response and IFN release. Explants from COPD patients and smokers was also exposed to two RV serotypes, RV-16 and RV-1B, in order to compare findings with a clinically relevant stimulant. Results: Poly(I:C) and R848 caused a significant increase of protein and gene expression of pro-inflammatory cytokines (TNFα, CCL5 and IL-6). Type I and III IFN gene expression was also significantly increased. Using the two ligands together caused a synergistic release of TNFα and CCL5. Tissue from COPD patients released more pro-inflammatory cytokines and expressed less IFNβ when compared to smokers. TNFα neutralisation inhibited subsequent release of CCL5 and IL-6. Dexamethasone and p38 MAPK inhibitor decreased TLR3- and TLR7/8-induced pro-inflammatory response whereas IKK-2 and IRAK1/4 inhibition had little effect on cytokine release. Dexamethasone and IKK-2 showed limited effect on IFN gene expression whereas p38 MAPK inhibitor significantly decreased and IRAK1/4 inhibition enhanced IFN expression. RV-16 induced modest but significant pro-inflammatory response in lung tissue, whereas RV-1B did not induce cytokine release. Both serotypes induced type I and III IFN gene expression. Tissue from COPD patients showed a lower expression of IFNβ and IFNλ when compared to smokers. Conclusion: This tissue explant was responsive to both synthetic TLR ligands and RV. The release of pro-inflammatory cytokines in response to TLR stimulation was partially inhibited by steroid. p38 MAPK is involved in TLR-induced inflammation but it also further decreases the already impaired IFN gene expression in COPD tissue. The role of IKK-2 and IRAK1/4 in TLR-induced innate immune response remains unclear. This model is a valuable system to study the mechanisms underlying RV-induced COPD exacerbations and also to test new inhibitors in the whole tissue environment.

Doctor of Philosophy / Department of Agronomy / Masaaki Tamura / Over the years proteins/peptides have evolved as promising therapeutic agents in the treatment of cancer. Considering the advantages of peptides such as their small size, ease of synthesis, tumor-penetrating ability and bio-compatibility, present report discusses proof of concept for 1. C1B5 peptide of protein kinase Cγ and a low dose of gemcitabine combination treatment for peritoneally disseminated pancreatic cancer and 2. dTAT peptide nanoparticles mediated gene (angiotensin II type 2 receptor gene) therapy for lung cancer. 1. A significant reduction in intraperitoneally (IP) transplanted pancreatic carcinoma growth was demonstrated with C1B5 peptide and gemcitabine co-treatment in an immunocompetent mouse model. Increased number of Granzyme B positive cells was observed in treated mice ascites, suggesting the involvement of immune response in tumor attenuation. The strong effect observed in combination treatment might be because of increase in lymphocyte recruitment by gemcitabine followed by C1B5 peptide mediated CD8+ T-cells or NK cells activation apart from direct cancer cell apoptosis. 2. To test dTAT peptide nanoparticles (dTAT NPs) mediated therapeutic gene delivery, luciferase reporter gene containing dTAT nanoparticles were synthesized (dTAT/pLUC/Ca2+). Synthesis conditions for nanoparticles were optimized based on dTAT/pLUC/Ca2+ nanoparticles transfection efficiency. With the optimized conditions, dTAT NPs containing AT2R, TRAIL or miR-34a pDNA (dTAT/pAT2R, dTAT/TRAIL or dTAT/miR- 34a) were synthesized. Therapeutic potential of these NPs was analyzed in lung adenocarcinoma containing mice by administering them intravenously (IV) or/and intratracheally (IV). Combination treatment with the IV injection of the new dTAT/pAT2R/Ca2+ formulation and the IT injection of the original dTAT/pAT2R/Ca2+ formulation is effective in attenuation of developed human bronchioloalveolar carcinoma in the SCID mouse lungs. Findings from the above mentioned studies have vital clinical relevance as it implies that peptides alone or when used as gene delivery systems may prove to be beneficial in the treatment of various stages of cancer.

Acute respiratory distress syndrome (ARDS) is associated with high mortality rates . The aim of treatment and ventilation is to improve oxygenation. Prone positioning improves oxygenation in patients with ARDS by shifting blood flow to undamaged or better ventilated regions of the lung. Critical care nurses follow the current guidelines with respect to prone positioning, which are mostly based on the medical aspects of the treatment. Prone positioning challenges nursing care of these patients. The research question that emerges is: Which nursing interventions during prone positioning will benefit the patient and reduce or eliminate complications? The purpose of this research is to do a systematic review in order to: Explore the evidence in support of the beneficial nursing interventions during prone positioning of ventilated patients and to develop evidence-based nursing guidelines with regard to the nursing process. The research design can be described as an exploratory, descriptive and retrospective systematic review. The population consisted of experimental study designs, as well as comparative, non-randomised and observational studies on nursing interventions during the prone positioning process. Selected studies included a population of adult or paediatric subjects who were ventilated and turned into the prone position, and the search strategy was restricted to articles published or translated into English. Studies that included animals or neonates were excluded from this review. The data collection process involved the systematic extraction of relevant data onto standardised data abstraction forms and the assessment of the methodological quality of each study. Data were summarised into evidence tables and data from randomised controlled trials were used for meta-analysis. There were thirteen randomised controlled trials, of which only seven could be included for quantitative analysis. Forty five clinical trials involving prone positioning were identified, with a total population of 2 148 patients. Outcomes that were measured, included oxygenation outcomes, responder and non-responder groups, haemodynamic outcomes, complications in the prone position, mortality, the length of sta y in the intensive care units and the total number of ventilated days. Prone positioning showed significant increases in the PaO2 and PaO2 / FiO2 ratio. The effect of the outcomes compared against the different ventilation, sedation, nutrition and positioning protocols had inconclusive results. Haemodynamic variables had insignificant increases in the prone position. Pulmonary artery wedge pressure (PAWP) however, did show a significant increase in the prone position. Complications related to prone positioning were insignificantly less than expected. Patients treated in the prone position were ventilated for an insignificantly shorter period of time, but had a longer ICU stay, although the results were also insignificant. The mortality of patients in the selected trials was 33.5%. Evidence gained from the selected studies could be used to develop nursing guidelines, despite inconclusive results related to some of the measured outcomes. / Dr. Elzabé Nel

Abstract The tumour suppressor gene TP53 is the most commonly mutated gene in human cancers. The protein it codes, p53, becomes activated as a response to stress signals. When activated, p53 binds to DNA and affects the transcription of its target genes. They then cause cell cycle arrest, DNA repair and/or induction of programmed cell death, thus preventing mutations and cancer. Specific mutations in TP53 are associated with exposure to certain carcinogens, such as polycyclic aromatic hydrocarbons (PAHs). These environmental chemical carcinogens are formed through incomplete combustion of organic material. Benzo(a)pyrene (BP) is commonly used as a model compound for PAH carcinogenesis. BP causes accumulation of p53, but the mechanism of accumulation is not known. The aim of this study was to gain more insight into the p53 protein in the first phases of PAH carcinogenesis in vivo in mouse, using BP as the model compound. Mice from the inbred C57BL/6 strain were treated topically or intraperitoneally with BP or were exposed to cigarette smoke inhalation. The amount of p53 protein was studied by immunoblotting, immunohistochemistry and immuno electron microscopy, and the mdm2, p21 and p19ARF proteins were studied by immunoblotting. The binding of BP to DNA was measured by synchronous fluorescence spectrophotometry. The p53 protein was induced in vivo in skin and lung after BP treatment and in lung after cigarette smoke treatment. An increase in p53 was associated with an increase in the amount of BP-DNA adducts. In skin, the induction of p53 was accompanied by induction of the p21 and mdm2 proteins, which are transcriptional targets of p53. This indicates that the in vivo induced p53 is a wild-type protein and functional. In lungs, the induction of p53 was accompanied by a decrease of mdm2 and an increase of p19ARF. These results confirm that BP is metabolized and binds to DNA in mouse tissues and indicate that BP-DNA adducts are the trigger for p53 protein induction. The in vivo regulation of the p53 protein is different in different tissues of C57BL/6 mouse.

Abstract Epidemiological and experimental studies indicate a causative role of viruses in malignancies. Recently, a link between bacterial infections and the development of cancer has been suggested. The purpose of this study was to evaluate the association between chlamydial infection and cancer. The association between C. trachomatis infection and cervix cancer was analysed in a prospective study. The presence of IgG antibodies to C. trachomatis and C. pneumoniae was determined from the serum samples of 182 Nordic women with invasive cervical carcinoma and 538 matched cancer-free controls by the microimmunofluorescence (MIF) method. Serum antibodies to C. trachomatis were associated with an increased risk for cervical squamous cell carcinoma (SCC) (OR 2.2, 95% CI 1.3-3.5), but not for cervical adenocarcinoma (OR 0.4, 95% CI 0.1-1.7). C. trachomatis serotype G was highly significantly associated with an increased risk for SCC (adjusted OR 6.6, 95% CI 1.6-27). The presence of serum IgG antibodies to more than one serotype of C. trachomatis, on the other hand, also increased the risk of SCC. The association between C. pneumoniae infection and lung cancer was analysed separately in men and women. C. pneumoniae-specific antibodies and immune complexes (IC) were analysed from 230 Finnish smoking males with lung cancer and their matched controls using serum samples collected before the lung cancer diagnosis. Suggestive chronic C. pneumoniae infection was associated with an increased risk for lung cancer (OR 1.6; 95% CI 1.0-2.3). The risk was increased especially in men younger than 60 years (OR 2.9; 95% CI 1.5-5.4), but not in the older age group (OR 0.9; 95% CI 0.5-1.6). Chlamydial antibodies and chlamydia-specific ICs were analysed from serum samples of 29 Finnish women with lung cancer and 87 matched cancer-free controls by MIF. The mean follow-up from serum sampling to cancer diagnosis was 6.7 years. IgG class antibodies to C. pneumoniae were common in pregnant Finnish women (66% among cases, 62% among controls), whereas IC-bound C. pneumoniae IgG antibodies were rare. No additional risk for lung cancer in association with chlamydial antibodies was found among women. The association between chlamydial infections and lymphomas was evaluated in a cross-sectional study. Seventy-two lymphoma patients from Tampere University Hospital and 72 matched controls were selected, and IgG antibodies and ICs to C. pneumoniae and C. trachomatis were analysed from their serum samples by MIF and enzyme immunoassay (EIA). The serological markers suggesting chronic chlamydial infection were associated with an increased risk for malignant lymphoma. The association was most evident for the presence of C. pneumoniae-specific ICs in non-Hodgkin's lymphoma (OR = 7.3, 95% CI 2.2-25) and appeared to be limited to men. Infection with C. trachomatis was found to increase the risk of subsequent development of invasive cervical SCC. Chronic C. pneumoniae infection was also found to be a new independent risk factor for lung cancer in males. Serological markers suggestive of chronic chlamydial infection were associated with lymphomas, proposing that chlamydial infection may have a similar role as H. pylori in the pathogenesis of lymphomas.

Abstract Cytochrome P450 2A6 (CYP2A6) is involved in the 7-hydroxylation of coumarin, C-oxidation of nicotine, and the metabolism of tobacco specific nitrosamines. Initially in 1995 Fernandez-Salguero et al. reported a genotyping method for three alleles: CYP2A6*1 (wild-type), CYP2A6*2 (variant 1), and CYP2A6*3 (variant 2). Later studies presented in this thesis indicated that the original genotyping method produces erroneous results for the CYP2A6*3 allele due to unspecific PCR conditions and previously unknown CYP2A6*1B allele. Furthermore, the CYP2A6*2 allele genotyping caused erroneous genotypes (CYP2A6*2/*2 was misclassified as CYP2A6*1/*2). In this work, new PCR based genotyping methods were developed for CYP2A6*2 and for several new alleles (CYP2A6*1B, CYP2A6*4A/*4D and CYP2A6*5). In population-based studies, the deletion alleles (pooled as CYP2A6*4) turned out to be more prevalent among Asians (15.1%) than Caucasians (0.5%). The frequencies of the other inactive alleles varied within 0–3% in both populations. Asians totally lacked the CYP2A6*2 allele, whereas Caucasians lacked the CYP2A6*5 allele. The frequencies of two wild-type alleles, CYP2A6*1A and CYP2A6*1B alleles were 66.5% and 30.0% in Caucasians, and 43.2% and 40.6% in Asians, respectively. Correlation studies between the phenotype, as tested by the administration of coumarin, and the genotype demonstrated that individuals with the CYP2A6*2/*2 genotype were totally defective, while CYP2A6*1/*2 subjects exhibited intermediate and CYP2A6*1/*1 subjects full capablility of producing 7-hydroxycoumarin. Upon phenotyping with nicotine, individuals with the CYP2A6*1/*2 or CYP2A6*1/*4 genotype were shown to have a lower enzyme activity (one fourth of the normal activity), compared to those with the CYP2A6*1/*1 genotype. Defective CYP2A6 activity has been hypothesised to reduce the risk of environmentally (especially tobacco smoke) induced diseases either by decreasing production of genotoxic metabolites or by preventing addiction to tobacco smoking. However, in our case-control studies on Spanish patients with liver cirrhosis (n = 83) and liver cancer (n = 90) and their controls (n = 237) no significant association between the CYP2A6 genotypes and disease proneness was found. The odds ratio (OR) for developing liver cancer was was 1.4 (95% confidence interval [CI] 0.5–3.7) for genotypes containing at least one CYP2A6*2 allele. For liver cancer the respective OR was 1.3 (95% CI 0.4–4.5). Similarly, no statistically association between CYP2A6 alleles and the risk of lung cancer was observed in our Finnish study population cinsisting of 177 cases and 1089 controls; the OR for combined CYP2A6 variant allele containing genotypes (CYP2A6*1/*2 and CYP2A6*1/*4) was 1.19 (95% CI 0.56–2.45). Our studies therefore do not indicate any major modifying role for the CYP2A6 genotypes in individual susceptibility to environmentally induced diseases.

Background: Lung function is a highly heritable trait. So far there is limited knowledge of the genetic factors that influence lung function. Vascular endothelial growth factor A (VEGF-A) is expressed in the lung at high levels and is known to play a role in angiogenesis and lung remodelling, both in utero and throughout life. A candidate gene study was carried out in order to investigate the role of variants within the VEGF-A gene in determining lung function in childhood and adult life.Methods: Using available longitudinal data previously collected for an unselected birth cohort (Manchester Asthma and Allergy Study-MAAS) the relationship between lung function and single-nucleotide polymorphisms (SNPs) in VEGF-A was assessed. Replication studies were performed in cross-sectional studies of adults from Manchester and children with asthma from Croatia, in whom FEV1/FVC ratio was measured using spirometry. The potential functional roles of two consistently associated SNPs were then further investigated. Finally, using the genome-wide data generated in the discovery cohort (MAAS) I assessed why associations between VEGF-A and lung function had not been reported in recent genome-wide association studies of lung function.Results: Two VEGF-A SNPs, rs10434 and rs3025028, were significantly associated with lung function at multiple ages in a discovery population (MAAS). Subjects with a GG genotype for either SNP had significantly diminished lung function compared to subjects with other genotypes. These findings were replicated in two additional populations (631 parents of children participating in MAAS and in 410 Croatian children with physician-diagnosed asthma aged 6-18 years). SNP rs10434 is located in the 3’UTR and based on its location I hypothesised that it may affect mRNA stability. No significant difference in the rate of VEGF-A mRNA degradation was found between GG and the AA homozygotes. SNP rs3025028 is an intronic SNP in a close proximity to the splice site involved in alternative splicing which generates two different isoforms of VEGF-A; I therefore tested the hypothesis that a change of base at this position could affect the splicing mechanism and cause a change in the ratio of the isoforms. Western blot analysis was used to demonstrate a difference in the ratio of the splice variants VEGF-A165b and total VEGF-A165 (relative to a reference sample) between genotype groups. The VEGF-A165b/panVEGF-A165 ratio was significantly higher at birth (cord plasma), in school-age children and in adults amongst CC compared to GG homozygotes at rs3025038 (p<0.03). Finally, the genome-wide data for the discovery cohort showed that the region containing VEGF-A was not well targeted by either genotyped or imputed SNPs in genome-wide arrays. Conclusion: Evidence was provided to demonstrate that variants within the VEGF-A gene are significantly associated with lung function in both children and adults. Furthermore, data was presented to support a functional role for one of the SNPs (rs3025028). I investigated why associations between VEGF-A and lung function had not previously been reported in recent GWAS and concluded that the region containing VEGF-A was poorly covered by all of the currently available arrays.