Evaluating Small Airways Disease in Asthma and COPD using the Forced Oscillation Technique and Magnetic Resonance Imaging

Obstructive lung disease, including asthma and chronic obstructive pulmonary disease (COPD), is characterized by heterogeneous ventilation. Unfortunately, the underlying structure-function relationships and the relationships between measurements of heterogeneity and patient quality-of-life in obstructive lung disease are not well understood. Hyperpolarized noble gas MRI is used to visualize and quantify ventilation distribution and the forced oscillation technique (FOT) applies a multi-frequency pressure oscillation at the mouth to measure respiratory impedance to airflow (including resistance and reactance). My objective was to use FOT, ventilation MRI and computational airway tree modeling to better understand ventilation heterogeneity in asthma and COPD. FOT-measured respiratory system impedance was correlated with MRI ventilation heterogeneity and both were related to quality-of-life in asthma and COPD. FOT-measurements and model-predictions of reactance and small-airways resistance were correlated in asthma and COPD respectively. This study is the first to demonstrate the relationships between FOT-measured impedance, MRI ventilation heterogeneity, and patient quality-of-life

3-D lung deformation and function from respiratory-gated 4-D x-ray CT images : application to radiation treatment planning.

Many lung diseases or injuries can cause biomechanical or material property changes that can alter lung function. While the mechanical changes associated with the change of the material properties originate at a regional level, they remain largely asymptomatic and are invisible to global measures of lung function until they have advanced significantly and have aggregated. In the realm of external beam radiation therapy of patients suffering from lung cancer, determination of patterns of pre- and post-treatment motion, and measures of regional and global lung elasticity and function are clinically relevant. In this dissertation, we demonstrate that 4-D CT derived ventilation images, including mechanical strain, provide an accurate and physiologically relevant assessment of regional pulmonary function which may be incorporated into the treatment planning process. Our contributions are as follows: (i) A new volumetric deformable image registration technique based on 3-D optical flow (MOFID) has been designed and implemented which permits the possibility of enforcing physical constraints on the numerical solutions for computing motion field from respiratory-gated 4-D CT thoracic images. The proposed optical flow framework is an accurate motion model for the thoracic CT registration problem. (ii) A large displacement landmark-base elastic registration method has been devised for thoracic CT volumetric image sets containing large deformations or changes, as encountered for example in registration of pre-treatment and post-treatment images or multi-modality registration. (iii) Based on deformation maps from MOFIO, a novel framework for regional quantification of mechanical strain as an index of lung functionality has been formulated for measurement of regional pulmonary function. (iv) In a cohort consisting of seven patients with non-small cell lung cancer, validation of physiologic accuracy of the 4-0 CT derived quantitative images including Jacobian metric of ventilation, Vjac, and principal strains, (V?1, V?2, V?3, has been performed through correlation of the derived measures with SPECT ventilation and perfusion scans. The statistical correlations with SPECT have shown that the maximum principal strain pulmonary function map derived from MOFIO, outperforms all previously established ventilation metrics from 40-CT. It is hypothesized that use of CT -derived ventilation images in the treatment planning process will help predict and prevent pulmonary toxicity due to radiation treatment. It is also hypothesized that measures of regional and global lung elasticity and function obtained during the course of treatment may be used to adapt radiation treatment. Having objective methods with which to assess pre-treatment global and regional lung function and biomechanical properties, the radiation treatment dose can potentially be escalated to improve tumor response and local control

Comparison of hyperpolarised gas MRI and CT-based surrogates of ventilation

Background: Non-contrast CT-based surrogates of regional ventilation derived from pulmonary images acquired at multiple inflation levels have been proposed as alternatives to established modalities. However, their physiological accuracy has yet to be validated prior to clinical translation. Purpose: To address the hypothesis that these surrogates can provide information comparable to a direct measure of ventilation from hyperpolarised gas MRI ventilation via: i. development of a methodology for registering CT and gas MRI. ii. comparison of these surrogates with gas MRI at the lobar level. iii. evaluation of the impact of inflation levels when comparing gas MRI and ventilation CT. iv. development of an image acquisition and analysis framework to facilitate spatial correlations of both techniques. v. assessment of the effect of using different gases on the correlation. Methods: i. A method to indirectly register gas MRI to CT via same-breath 1H-structural MR images was developed and its accuracy was assessed. ii. A ventilation model based on expansion of lobar CT segmentations was compared with gas MRI lobar ventilation measurements. iii. The spatial overlap of ventilation CT was compared to gas MRI acquired at two different inflation levels. iv. An image acquisition protocol was designed to minimise differences in acquisition settings between scans such as posture and breathing manoeuvre and analysis methods were developed to enable direct regional and voxel level correlations. v. The effect of using two different noble gases, namely, 3He and 129Xe, on correlation with ventilation CT was assessed. Results: i. The indirect method of registration was more accurate than direct registration. ii. Despite subtle differences, lobar ventilation measurements derived from CT and hyperpolarised gas MRI were comparable. iii. Comparison of ventilation CT and gas MRI varied with inflation state. iv. The spatial correlation between ventilation CT and gas MRI increased at coarser levels. v. A marked improvement in correlation was observed for 3He and 129Xe MRI in contrast to when ventilation CT was compared with either 3He and 129Xe MRI. Conclusion: Although CT-based surrogates of ventilation show promise for replacing established ventilation modalities such as hyperpolarised gas MRI, particularly at coarser levels, they cannot be assumed to be equivalent to the techniques they purport to replace

Towards a non-invasive diagnostic aid for abdominal adhesions using dynamic MRI and image processing

This work presents a strategy for detection of abdominal adhesions based on cine-MRI data, image processing and the production of a ‘sheargram’. Abdominal adhesions are a common complication of abdominal surgery and can cause serious morbidity. Diagnosis is difficult and often one of exclusion. A conclusive diagnosis typically requires laparoscopic explorative surgery, which itself may cause further adhesions. A non-invasive means of diagnosis is preferred and likely to aid patient management. Cine-MRI can capture the motion of the abdominal structures during respiration and has shown promise for adhesion detection. However, such images are difficult and time consuming to interpret. A previous PhD considered augmenting cine-MRI by quantifying movement for detection of gross adhesive pathology. This thesis presents a refined image processing approach aimed at detection of more subtle adhesions to the abdominal wall. In the absence of adhesive pathology, the abdominal contents (bowels, kidneys, liver) slide smoothly against the perimeter of the abdominal cavity – a process termed visceral slide. An adhesion is expected to produce a localised resistance that inhibits smooth visceral sliding. In this PhD, development of a 2D technique to quantify sliding around the perimeter of the abdominal cavity (with particular emphasis on the abdominal wall) sought to highlight regions of reduced sliding. Segmentation and image registration were employed to quantify movement and shear, the latter used as an analogue for sliding. The magnitude of shear over all frames in the dynamic MR image sequence was extracted and displayed as a colour plot over the MR image for anatomical context. This final output is termed a ‘sheargram’. Suitability of the technique for diagnosis was assessed through a series of experimental tests and correlation with clinical data. The latter involved a retrospective pilot study incorporating data from 52 patients scanned for suspected adhesions. A total of 141 slices were processed and reported. The validation experiments confirmed the technique had the attributes to accurately and reproducibly report sliding and demonstrated proof of concept for detection of adhered regions. The pilot study confirmed the sheargram matched expert clinical judgement in the vast majority of cases (>84%) and detected >93% of all adhesions. However, the investigation also highlighted limitations, principally structures moving out of the imaging plane creates a fundamental problem and requires a 3D imaging solution. In conclusion, the work has produced encouraging results and merits further development

The role of microRNA in the development of pulmonary arterial hypertension: studies in cell culture and animal models.

Pulmonary arterial hypertension (PAH) is a complex disease characterised by narrowing and remodelling of the small pulmonary arteries. This process involves all cell types within the vessel wall and results in an increase in pulmonary artery pressure, right heart failure and can eventually lead to premature death. Diagnosis of PAH occurs late in disease progression with patients already displaying severe hemodynamic compromise and mortality rates remain unacceptably high despite current treatment. Therefore the development of new therapies is required to manage the symptoms and treat the underlying causes of this multifaceted disease. Recent studies have highlighted a role for microRNAs (miRNAs) in the initiation, development and progression of PAH. MiRNAs are small non-coding RNA molecules ~22 nucleotides long that negatively regulate gene expression. Previous work from our laboratory has shown that miRNAs are dysregulated within the lung during the development of experimental pulmonary hypertension (PH). Consequently, the aim of this study was to assess the involvement of specific miRNAs in the development of PAH using cell culture and experimental models of PH. The first miRNA focused on was miR-451 which is up-regulated in the lungs from animal models of PH. In human pulmonary artery smooth muscle cells (hPASMCs), miR-451 over-expression promoted migration in the absence of serum but had no effect on cellular proliferation. Silencing of miR-451 was performed in vivo using antimiR-451 and miR-451 knockout mice. Indices of PAH were assessed after exposure to hypoxia via measurement of right ventricular pressure (RVP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. There was a reduction in systolic RVP in hypoxic rats pre-treated with antimiR-451 compared to control antimiR (47.7 ± 1.36 mmHg and 56.0 ± 2.03 mmHg respectively, p<0.01). MiR-451 knockout mice exposed to chronic hypoxia displayed no significant differences for PAH indices compared to wild type hypoxic mice. Thus illustrating that transient inhibition of miR-451 attenuates the development of PH in hypoxic rats however, genetic deletion of miR-451 has no beneficial effect on the development of PH. This may be due to compensatory mechanisms present in the miR-451 knockout mice. Previous work has also shown that miR-145 is up-regulated in the lungs and pulmonary arteries from animal models of PH as well as PAH patients. Therefore miR-145 expression was modulated in rats using antimiR-145 both prior to and post exposure to hypoxia and SU5416 administration. Prophylactic silencing of miR-145 in the hypoxia/SU5416 model of PH showed no beneficial effect on the development of PH compared to control antimiR treated rats exposed to hypoxia. Therapeutic modulation of miR-145 also demonstrated no protective effect on RVP, RVH or muscularisation of pulmonary arteries in the rat hypoxia/SU5416 model. There was however a significant reduction in the number of occluded vessels in rats with established PH treated with antimiR-145. This reduction in occluded vessel count is interesting as it was not observed in the prevention study. Further work is required to pinpoint the exact mechanisms through which antimiR-145 is producing this positive effect on pulmonary vessels with therapeutic silencing of miR-145. The role of miR-145 on PAH development was further investigated with the use of miR-145 knockout mice. Recent studies show that genetic ablation of miR-145 protects female mice from developing hypoxia-induced PH. We therefore sought to establish whether this beneficial response was also observed in male miR-145 knockout mice. Hypoxic male miR-145 knockout mice showed similar indices of PAH as hypoxic miR-145 wild type mice, with increased RVP and RVH compared with normoxic mice. Pulmonary vascular remodelling analysis indicates that miR-145 knockout mice exposed to hypoxia may have a reduction in remodelling compared to wild type hypoxic mice however this does not reach significance. Thus it appears from this study that male miR-145 knockout mice are not protected against developing PAH as the female knockout mice are. The results from this study on male miR-145 knockout mice demonstrate that the effects of silencing miR-145 in vivo are indeed gender specific. As well as affecting the pulmonary arteries, PAH also induces changes within the right ventricle culminating in right ventricular dysfunction and failure. Therefore a miRNA profile was established for the PAH diseased right ventricle. MiR-27a and miR-27b were up-regulated within the right ventricle of hypoxia/SU5416 mice and rats, respectively. This response appears to be cardiac specific and may help to establish therapies to maintain and stabilise RV function. In summary of these findings, we have confirmed that miRNAs are dysregulated within the lung and right ventricle during PH development. Results suggest that there are complex mechanisms regulating miRNA processing within the lung during the development of PAH and that these pathways may be gender specific. Further work is required to understand the genes targeted, and therefore the pathways modulated, by miRNAs during PAH development to enhance our understanding of the intricate systems involved in disease progression. MiRNAs represent a potential therapeutic target for the treatment of PAH with further work required to pinpoint the exact mechanistic pathways through which they exert their effects

The role of non-coding RNA in the development of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease of the small pulmonary arteries, characterised by pulmonary vascular remodelling due to excessive proliferation and resistance to apoptosis of pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs). The increased pulmonary vascular resistance and elevated pulmonary artery pressures result in right heart failure and premature death. Germline mutations of the bone morphogenetic protein receptor-2 (bmpr2) gene, a receptor of the transforming growth factor beta (TGF-β) superfamily, account for approximately 75%-80% of the cases of heritable form of PAH (HPAH) and 20% of sporadic cases or idiopathic PAH (IPAH). IPAH patients without known bmpr2 mutations show reduced expression of BMPR2. However only ~ 20% of bmpr2-mutation carriers will develop the disease, due to an incomplete penetrance, thus the need for a ‘second hit’ including other genetic and/or environmental factors is accepted. Diagnosis of PAH occurs most frequently when patients have reached an advanced stage of disease. Although modern PAH therapies can markedly improve a patient’s symptoms and slow the rate of clinical deterioration, the mortality rate from PAH remains unacceptably high. Therefore, the development of novel therapeutic approaches is required for the treatment of this multifaceted disease. Noncoding RNAs (ncRNAs) include microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). MiRNAs are ~ 22 nucleotide long and act as negative regulators of gene ex-pression via degradation or translational inhibition of their target mRNAs. Previous studies showed extensive evidence for the role of miRNAs in the development of PAH. LncRNAs are transcribed RNA molecules greater than 200 nucleotides in length. Similar to classical mRNA, lncRNAs are translated by RNA polymerase II and are generally alternatively spliced and polyadenylated. LncRNAs are highly versatile and function to regulate gene expression by diverse mechanisms. Unlike miRNAs, which exhibit well-defined actions in negatively regulating gene expression via the 3’-UTR of mRNAs, lncRNAs play more diverse and unpredictable regulatory roles. Although a number of lncRNAs have been intensively investigated in the cancer field, studies of the role of lncRNAs in vascular diseases such as PAH are still at a very early stage. The aim of this study was to investigate the involvement of specific ncRNAs in the development of PAH using experimental animal models and cell culture. The first ncRNA we focused on was miR-143, which is up-regulated in the lung and right ventricle tissues of various animal models of PH, as well as in the lungs and PASMCs of PAH patients. We show that genetic ablation of miR-143 is protective against the development of chronic hypoxia induced PH in mice, assessed via measurement of right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. We further report that knockdown of miR-143-3p in WT mice via anti-miR-143-3p administration prior to exposure of mice to chronic hypoxia significantly decreases certain indices of PH (RVSP) although no significant changes in RVH and pulmo-nary vascular remodelling were observed. However, a reversal study using antimiR-143-3p treatment to modulate miR-143-3p demonstrated a protective effect on RVSP, RVH, and muscularisation of pulmonary arteries in the mouse chronic hypoxia induced PH model. In vitro experiments showed that miR-143-3p overexpression promotes PASMC migration and inhibits PASMC apoptosis, while knockdown miR-143-3p elicits the opposite effect, with no effects observed on cellular proliferation. Interestingly, miR-143-3p-enriched exosomes derived from PASMCs mediated cell-to-cell communication between PASMCs and PAECs, contributing to the pro-migratory and pro-angiogenic phenotype of PAECs that underlies the pathogenesis of PAH. Previous work has shown that miR-145-5p expression is upregulated in the chronic hypoxia induced mouse model of PH, as well as in PAH patients. Genetic ablation and pharmacological inhibition (subcutaneous injection) of miR-145-5p exert a protective against the de-velopment of PAH. In order to explore the potential for alternative, more lung targeted delivery strategies, miR-145-5p expression was inhibited in WT mice using intranasal-delivered antimiR-145-5p both prior to and post exposure to chronic hypoxia. The decreased expression of miR-145-5p in lung showed no beneficial effect on the development of PH compared with control antimiRNA treated mice exposed to chronic hypoxia. Thus, miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while the inhibition of miR-143-3p prevented the development of experimental pulmonary hypertension. We focused on two lncRNAs in this project: Myocardin-induced Smooth Muscle Long noncoding RNA, Inducer of Differentiation (MYOSLID) and non-annotated Myolnc16, which were identified from RNA sequencing studies in human coronary artery smooth muscle cells (HCASMCs) that overexpress myocardin. MYOSLID was significantly in-creased in PASMCs from patients with IPAH compared to healthy controls and increased in circulating endothelial progenitor cells (EPCs) from bmpr2 mutant PAH patients. Exposure of PASMCs to hypoxia in vitro led to a significant upregulation in MYOSLID expres-sion. MYOSLID expression was also induced by treatment of PASMC with BMP4, TGF-β and PDGF, which are known to be triggers of PAH in vitro. Small interfering RNA (siR-NA)-mediated knockdown MYOSLID inhibited migration and induced cell apoptosis without affecting cell proliferation and upregulated several genes in the BMP pathway in-cluding bmpr1α, bmpr2, id1, and id3. Modulation of MYOSLID also affected expression of BMPR2 at the protein level. In addition, MYOSLID knockdown affected the BMP-Smad and BMP-non-Smad signalling pathways in PASMCs assessed by phosphorylation of Smad1/5/9 and ERK1/2, respectively. In PAECs, MYOSLID expression was also induced by hypoxia exposure, VEGF and FGF2 treatment. In addition, MYOSLID knockdown sig-nificantly decreased the proliferation of PAECs. Thus, MYOSLID may be a novel modulator in pulmonary vascular cell functions, likely through the BMP-Smad and –non-Smad pathways. Treatment of PASMCs with inflammatory cytokines (IL-1 and TNF-α) significantly in-duced the expression of Myolnc16 at a very early time point. Knockdown of Myolnc16 in vitro decreased the expression of il-6, and upregulated the expression of il-1 and il-8 in PASMCs. Moreover, the expression levels of chemokines (cxcl1, cxcl6 and cxcl8) were sig-nificantly decreased with Myolnc16 knockdown. In addition, Myolnc16 knockdown decreased the MAP kinase signalling pathway assessed by phosphorylation of ERK1/2 and p38 MAPK and inhibited cell migration and proliferation in PASMCs. Thus, Myolnc16 may a novel modulator of PASMCs functions through anti-inflammatory signalling pathways. In summary, in this thesis we have demonstrated how miR-143-3p plays a protective role in the development of PH both in vivo animal models and patients, as well as in vitro cell cul-ture. Moreover, we have showed the role of two novel lncRNAs in pulmonary vascular cells. These ncRNAs represent potential novel therapeutic targets for the treatment of PAH with further work addressing to investigate the target genes, and the pathways modulated by these ncRNAs during the development of PAH