Donald Sin

Professor

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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Intratracheal lipopolysaccharide exposure induced atherosclerotic plaque destabilization : a novel murine plaque vulnerability model (2016)

Rationale: Literature has implicated lung inflammation as a risk factor for acute cardiovascular (CV) diseases (CVD), but the underlying mechanisms linking lung injury with CVD are largely unknown. Plasma neutrophilic myeloperoxidase (MPO) is an inflammatory biomarker for acute CVD, but its pathological role in CVD is unclear.Hypothesis: A murine model of atheromatous plaque vulnerability can be established by intratracheal Lipopolysaccharide (LPS) exposure. Neutrophils and MPO may contribute to this process.Approaches: LPS (3 mg/kg) or saline (control) was instilled directly into the lungs of male apolipoprotein E-knockout (ApoE-/-) C57BL/6J mice following 8 weeks of a Western-type diet. 24 hours later, atheromas in the right brachiocephalic trunk (BCT) were assessed for stability ex vivo using high-resolution optical projection tomography (OPT) and histology. Circulating neutrophils were depleted in vivo using neutrophil-specific antibodies to investigate the role of neutrophils in this model and determine the source of MPO. 4-aminobenzoic acid hydrazide (4-ABAH) was injected intraperitoneally to inhibit MPO. Results: LPS-exposed mice developed vulnerable plaques, characterized by intraplaque hemorrhage and thrombus compared to saline-exposed mice (p=0.0004). Plaque vulnerability was detectable as early as 8 hours post-intratracheal LPS instillation but not with intraperitoneal instillation. Depletion of circulating neutrophils attenuated plaque destabilization (P=0.027). MPO was found acutely localized in the vulnerable plaques and attached thrombi of LPS-exposed mice but not in the stable plaques of saline-exposed mice. Enzymatic inhibition of MPO in LPS-exposed mice decreased plaque vulnerability (P=0.038) and MPO/HOCl mediated oxidation (P=0.0076) Depletion of circulating neutrophils in LPS-exposed mice prevented intraplaque MPO accumulation, confirming that they were the major source of MPO in this model.Conclusion: We have established a novel plaque vulnerability model related to lung inflammation induced by intratracheal exposure to LPS. In this model, neutrophils play an important role in both lung inflammation and plaque destabilization. 3D OPT analysis revealed that during LPS-induced lung inflammation, MPO localizes acutely in atherosclerotic plaques and contributes to plaque vulnerability. MPO could be an important therapeutic target for prevention of acute CV events related to lung injury. This model could be useful for screening therapeutic targets to prevent acute vascular events related to lung inflammation.

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The impact of female sex hormones on cigarette smoke-induced airway remodelling and mucus production (2016)

Adjusting for amount of smoking, women have a 50% increased risk of COPD compared with men. It is not known what the anatomic basis/mechanism(s) of these sex-related differences in COPD might be. The main objective of this study is to characterize the impact of female sex hormones on chronic cigarette smoke-induced airway remodelling and emphysema in a murine model of COPD. We showed here for the first time that smoke-induced COPD in female compared to male mice have increased small airway remodelling, and may be biologically driven by estrogen through down-regulation of antioxidant defences and activation of TGFβ1 signalling, resulting in increased expression of collagen matrix in the airway walls. These effects can be ameliorated by ovariectomy before smoke exposure or use of the estrogen antagonist, tamoxifen, during smoke exposure, suggesting that estrogen is involved in this process. Using the flexiVent system to assess the functional relationship with the observed structural changes, we showed evidence of cigarette smoke-induced lung abnormalities. Tissue damping (G), and complex input resistance of the respiratory system (Zrs) at low oscillating frequency were elevated in female compared to male mice after smoke exposure, and this effect was attenuated after ovariectomy. Quasistatic pressure-volume curve revealed a decrease in inspiratory capacity in female mice but not in male mice after smoke exposure, and this effect was attenuated after ovariectomy. Chronic smoke exposure did not increase goblet cell expression in the distal airways of all groups, suggesting that the increase in distal airway resistance in smoke-exposed female mice is unlikely to be derived from luminal exudates.Finally, using a human bronchial epithelial cell culture model in air liquid interface, we showed that transfection with nuclear factor of activated T-cell (NFAT)c1 or NFATc2 siRNA blunted estrogen or progesterone-induced increase in MUC5AC mRNA expression, respectively.Collectively, our data showed that estrogen may be involved in the excess risk for small airways disease in a mouse model of COPD, and MUC5AC expression is regulated by estrogen and progesterone via NFATc1 and NFATc2 in normal human bronchial epithelial cells.

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The bacterial lung tissue microbiome in the pathogenesis of chronic obstructive pulmonary disease (2015)

Rationale: Several laboratories have shown that the decline in lung function in Chronic Obstructive Pulmonary Disease (COPD) is associated with increased formation of tertiary lymphoid follicles. This provides direct histological evidence in support of the hypothesis that the decline in lung function is associated with activation of an adaptive immune response. The antigens responsible for driving this immune activation remain poorly understood. The recent realization that the human lung contains a bacterial microbiome that changes in association with the presence of COPD suggests the hypothesis that bacteria arising from within this microbiome might be responsible for activating the adaptive immune response in COPD. Approach: The research described in this thesis examines the lung tissue bacterial microbiome from patients with mild to moderate COPD as well as patients with very severe COPD. The bacterial microbiome from these studies utilized either nested or touchdown PCR followed by 454™ pyrotag sequencing of specific variable regions on the 16S rRNA gene. Changes in the microbiome were examined in relation to histological estimates of emphysematous destruction of the lung and inflammatory immune cell infiltration associated with this tissue remodeling process. Finally, Haemophilus influenzae, a bacterium identified from this microbiome, known to cause inflammation was compared to the host tissue repair process. Results: The different bacterial community was present in control and mild (GOLD 1) compared to moderate (GOLD 2) COPD. The community composition was also different between donor lung tissue and very severe (GOLD 4) COPD. Further, the analysis identified a list of 10 OTUs that discriminated between lung tissue affected by GOLD 4 COPD and controls. In addition, the data presented here indicate that the host immune response to these organisms precedes the structural changes associated with COPD. Conclusion: Collectively, these data confirm that there is a small but diverse microbiome in the normal human lung that becomes less diverse in COPD. Furthermore, the disappearance/appearance of certain OTUs can discriminate between control and COPD affected lung tissue and that some of these OTUs are associated with the inflammatory immune cell infiltration and tissue destruction that occurs in COPD.

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Master's Student Supervision (2010 - 2018)
Microbiota and telomere shortening in gut-lung axis of human immunodeficiency virus infected donors (2018)

Previous studies have demonstrated the existence of a gut-lung axis and its proposed role in altering immune response and respiratory disease pathogenesis. The lung and gut microbiomes have been individually studied in the context of Human Immunodeficiency Virus (HIV) infection; however the gut-lung axis in HIV remains relatively unexplored and requires further investigation. This study examined the microbiomes of the gut-lung axis and telomere attrition in HIV subjects using paired human lung and small intestine tissue.Paired lung and small intestine tissue autopsy specimens were obtained from ART-treated HIV-positive and HIV-negative donors through the National NeuroAIDS Tissue Consortium. Frozen samples were sectioned on dry ice and weighed for DNA extraction using the QIAGEN DNeasy® Blood and Tissue kit. Droplet digital PCR was used to measure bacterial load and quantitative PCR was performed to determine absolute telomere length (aTL). The 16S rRNA V4 region was sequenced using the MiSeq sequencing platform. Extraction negatives were run through the entire workflow alongside samples for quality control. Raw sequence reads were processed using QIIME2 (Quantitative Insights Into Microbial Ecology 2, v2018.2) followed by statistical analysis using R studio. Several t-tests, linear regression, PERMANOVA and ANCOVA were used for statistical analysis.Microbial composition was not found to differ significantly between ART-treated HIV-positive and HIV-negative donors in both lung and small intestine. However, bacterial load and abundance of individual amplicon sequence variants (ASVs) were found to be correlated in the lung and small intestine. Bacteroides was decreased in ART-treated HIV-positive donors and identified as one of the bacteria most likely to help explain differences between HIV-negative and ART-treated HIV-positive microbiomes. Telomere length was demonstrated to be shortened in lungs of ART-treated HIV-positive donors in comparison to HIV-negative but no differences were found between the two groups in the small intestine.ART-treated HIV donors present microbiomes similar to HIV-negative donors, suggesting that ART can provide the microenvironment necessary to maintain a “healthy” microbiome. Furthermore, microbiomes of the gut-lung axis have demonstrated a correlation in bacterial load and abundance of individuals ASVs, suggesting a strong relationship between the two sites in both ART-treated HIV donors and HIV-negative donors.

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An exploration of the lung microbiome and DNA methylation in patients infected with human immunodeficiency virus (2016)

Background: With the advent of antiretroviral therapy, patients infected with human immunodeficiency virus (HIV) can achieve near normal life expectancies. However, the risk for chronic illnesses such as chronic obstructive pulmonary disease (COPD) remains higher in HIV-infected patients despite improved survival. In disease states such as COPD, the microenvironment of the lungs can change dramatically, which creates permissive niches that allow for selective growth and reproduction of microbes. These changes may influence or be influenced by epigenetic alterations, specifically the methylation and demethylation of sites along the genetic code. We hypothesize that the microbiome and methylation profiles in the lower airway of HIV-infected patients are different compared to those of uninfected patients. These changes may prove relevant to the progression of chronic lung complications.Experimental Approach: This thesis examined small airway epithelial cells collected from patients infected with HIV and from uninfected subjects. Data on bacterial microbiome were obtained by using touchdown polymerase chain reaction (PCR) on epithelial cell DNA followed by MiSeq sequencing of specific variable regions on the bacterial 16S rRNA gene. For methylation profiling, DNA samples from the same airway epithelial cells were bisulfite converted and sequenced with the Illumina Infinium 450K HumanMethylation platform. Microbial diversity calculations, and linear regression models of methylation data were analyzed on Vegan and Limma packages in R, respectively.Results: The bacterial microbiome from small airway epithelial cells within the lungs of HIV-infected patients was distinct from that in uninfected patients. These changes included decreased diversity and decreased richness. Moreover, certain operational taxonomic units (OTUs) were able to distinguish HIV from non-HIV samples. Furthermore, a significant difference in global methylation patterns was identified.Conclusion: The microbiome and methylation profiles from small airway epithelial cells in HIV-infected individuals are significantly different from those in uninfected individuals, which may in part explain the heightened susceptibility of HIV-infected patients to COPD.

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Biomarkers for acute exacerbation of chronic obstructive pulmonary disease (2016)

Rationale: There are currently no generally accepted and validated blood tests available for diagnosing acute exacerbations of chronic obstructive pulmonary disease (AECOPD). There is an urgent need of biomarkers that can guide therapeutic management in AECOPD. Based on literature review, systemic inflammation and mild cardiac dysfunction are often associated with AECOPD. We hypothesized that certain protein markers can indeed be useful in tracking and diagnosing AECOPD progression.Methods: The study cohort consisted of 368 patients recruited in the chronic obstructive pulmonary disease (COPD) Rapid Transition Program who were hospitalized with a primary diagnosis of AECOPD, and 76 stable COPD patients who served as controls. We first determined the relationship of AECOPD of C-reactive protein (CRP) and the N-terminal of the prohormone brain natriuretic peptide (NT-proBNP). We then performed a discriminatory analysis using receiver-operating characteristics (ROC) curve in a logistic regression model. We compared the area under the curve (AUC) of 4 different combinations of CRP and NT-proBNP models. Lastly, we examined several potential biomarkers that were implicated in AECOPD.Results: The demographic data of the cohort and the controls were well matched, with an average age of 68 versus 65 years old, 64% versus 77% male, and a forced expiratory volume in 1 second (FEV1) % predicted of 52% versus 58%. The CRP and NT-proBNP levels at exacerbation onset were found to be the highest and progressively decreased over time. Of the 4 models of ROC curves, the leave-one-out cross-validated model including both CRP and NT-proBNP had an AUC of 0.80. This model replicated well in an external LEUKO dataset. On the iiother hand, D-Dimer, pulmonary and activation-regulated chemokine (PARC) and troponin I, showed minimal or no temporal changes during hospitalization and were no different than those with stable COPD.Conclusions: In summary, this thesis demonstrated that biomarkers such as CRP and NT-proBNP are significantly elevated during AECOPD and decreased with recovery. Secondly, a combination of CRP and NT-proBNP could discriminate patients who were hospitalized for their AECOPD from stable patients. Together, these two biomarkers show promise in diagnosing and tracking AECOPD.

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HIV-associated COPD : an exploration of risk factors and impacts (2014)

With recent advances in highly active antiretroviral therapy (HAART) and HIV-patient care, the longevity of individuals living with human immunodeficiency virus (HIV) infection has drastically increased. However, long term complications like chronic obstructive pulmonary disease (COPD) are synchronously more prevalent among this population. The fact that clinical manifestations of COPD develop in young HIV-infected individuals (20 to 50 years of age) compared older uninfected smokers (>50 years of age) suggests HIV-associated COPD may reflect a state of accelerated aging. In this thesis, we presented the clinical and HIV-related variables associated with telomere length, a measure of cellular age, in an HIV-infected cohort and explored the variables that determine poorer respiratory related health quality in this cohort. By using a novel technique to determine telomere length in peripheral leukocytes of HIV-infected and uninfected male subjects, we found that: firstly, telomere lengths were significantly shorter with HIV-infection even after adjusting for age, BMI, and smoking; secondly, telomere lengths were significantly and independently associated with age, FEV1(% of predicted), and soluble cluster of differentiation (sCD14); and thirdly, shorter telomere lengths were seen with worse emphysema scores as enumerated by computed tomography (CT) scans. Furthermore, the advantage of this new telomere length assay allowed us to determine that participants in our study demonstrated a “telomere age” roughly 40 years older than their biologic age, yet their respective slopes of decline with age remained parallel, when compared to an uninfected cohort. The St. George’s Respiratory Questionnaire was used to assess the influence of respiratory symptoms on a patient’s self-perceived health, social, and psychological status. Strikingly, we found HIV-infected patients are plagued with considerable respiratory limitations when compared to uninfected and even COPD counterparts. Also, we found that FEV1%pred, CD4 cell count, interleukin-6 (IL-6), but not telomere length were associated with worse respiratory health.

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Accelerated aging in COPD : the relationship of telomere length and mortality in COPD (2012)

The evidence for the role of accelerated aging in COPD progression is growing and the senescence hypothesis is one possible molecular pathway by which COPD develops. Telomeres are used as a biomarker of cellular aging, and cellular aging is accelerated by the presence of oxidative stress and inflammation. Previous studies have shown that telomeres of peripheral blood cells are significantly shorter in COPD patients, but no studies to date explored the relationship of telomere length to important health outcomes such as mortality. Using samples from Lung Health Study (LHS), we examined the role of telomere length and polymorphisms in genes involved in aging process in health outcomes in COPD patients. There were no significant differences in age, sex, BMI, race of cumulative smoking exposure (pack-years) among 4 groups, divided on basis of telomere length. However, the risk of all cause mortality was similar across the first 3 quartiles (short telomere) but dropped significantly in the 4th quartile (longest telomere, hazard ratio (HR), 1.30). Compared to individuals in the 4th quartile of relative telomere length, the remaining participants had significantly higher risk of cancer mortality (HR, 1.48). Smoking status did not make a significant difference in leukocyte telomere length but when compared to non-COPD, age matched control group, all smoker groups in LHS had shorter telomeres. We also investigated the role of SNPs that were previously associated with leukocyte telomere length in disease outcome. Although no SNPs were associated with leukocyte telomere length, several SNPs of telomere biology genes were associated with cardiovascular and lung cancer mortality. The rate of telomere attrition is influenced by both extrinsic factors, such as inflammation and oxidative stress and intrinsic factors such as genetic predisposition. Here, we have shown that accelerated aging of peripheral blood cells, indicated by short telomeres, seems to play a role in disease outcome of COPD. Although we failed to show significant associations between leukocyte telomere length and genetic polymorphisms of telomere regulatory genes, the SNPs may contribute to risk of mortality. Further research is needed to elucidate the pathways underlying these observations.

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Discovering inflammatory biomarkers in chronic obstructive pulmonary disease and cystic fibrosis : a case study of granzyme B (2011)

Granzymes, and particularly granzyme B (GzmB), are classically known to be involved in cell-mediated immunity and the induction of apoptosis through cell-specific targeting activity of cytotoxic T-lymphocytes in conjunction with perforin. However, recent literature has emerged that describes a largely overlooked role for GzmB in potentially mediating disease progression. This pathogenic role has been based on findings that GzmB can cleave extracellular matrix proteins while functioning independently of perforin. In chronic inflammatory lung states such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), there are important implications including the degradation of extracellular matrix leading to airway remodelling, reduced lung integrity, and emphysema. The generation of autoantigens may also result from cleavage of extracellular matrix proteins, contributing to inflammation. We measured GzmB levels in plasma and lung tissue homogenates of COPD subjects by enzyme-linked immunosorbent assays (ELISAs) to determine the relationship with lung function, measured by FEV₁% predicted and FEV₁/FVC ratio, and clinical COPD severity. We found that GzmB levels in the lung were positively associated with lung function. The data raise the possibility that the GzmB we measured may be part of a protective inflammatory response in the microenvironment, or it may be pathogenic in the early but not the later phases of COPD. In CF subjects, we measured levels of GzmB and other inflammatory biomarkers in plasma samples to determine their relationship with lung function parameters and hospitalization status. While plasma levels of GzmB were not related to lung function or hospitalization status, we found that IL-6, IL-1β, and LPS levels were significantly higher in hospitalized patients, and CRP, IL-6, IL-1β, and LBP were significantly correlated with lung function impairment. The results provide evidence that systemic inflammation is an independent factor associated with disease progression in CF and suggests an important role for chronic bacterial colonization in the lungs. Further research is needed to validate the pathogenic contributions of GzmB in diseases with chronic inflammatory lung states and to delineate the mechanisms of such potential contributions.

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