1,721,232 research outputs found
Effects of intermittent hypoxia and hyperlipidemia-in vivo and in vitro studies on pathogenetic mechanisms linking obstructive sleepapnea to cardiovascular disease
No full textpublished_or_final_versionMedicineDoctoralDoctor of Philosoph
Effects of human mesenchymal stem cells on cigarette smoke-induced lung damage
No full textChronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by persistent airway obstruction that is only partially reversible. It is the fourth leading cause of death and is predicted to be the third by 2030. The progression of the disease involves chronic inflammation, oxidative stress, excess protease activity, increased lung cell apoptosis and accelerated lung aging, but the exact pathogenesis is still unclear. The major cause of COPD is cigarette smoking(CS). Although COPD is associated with increasing social and economical burden, there have been few advances in pharmacological therapy of COPD.
Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells which can be isolated from a broad range of sources including bone marrow (BM) and adipose tissue. Administration of BM-derivedMSCs (BM-MSC) or adipose tissue-derived MSCs was reported to attenuate CS-induced emphysema in murine models. Induced pluripotent stem cell-derived MSC (IPSC-MSC) are MSCs differentiated from induced pluripotent stem cells(IPSCs), which are pluripotent cells generated by somatic cell reprogramming in vitro. IPSC-MSCs have several advantages over BM-MSC, including more abundant sources and high capacity of doubling without loss of differentiation potency.
A general exploration and comparison on the effects of human IPSC-MSC and BM-MSC treatments were carried out in a 56-day CS-exposed rat model. Compared to BM-MSC, IPSC-MSC showed a higher capacity to reside in lung tissue. The two treatments shared similar efficacy to attenuate CS-induced lung cell apoptosis, to restore CS-induced reduction of lungIL-10and to alleviate CS-induced elevation of systemic TGF-β1. In addition, IPSC-MSC was found to cause reduction in CS-induced elevation of systemic oxidative stress and reversal of CS-induced reduction of lung adiponectin.
Furthermore, in order to understand the possible paracrine mechanism involved, human airway epithelial cells were treated with IPSC-MSC or BM-MSC-conditioned medium in a cell culture system in the presence of cigarette smoke medium (CSM). Potentiation rather than attenuation of CSM-induced release of pro-inflammatory cytokine IL-8, MCP-1 and IL-6 was observed with IPSC-MSC or BM-MSC conditioned medium. It is currently unknown whether cultured IPSC-MSCs or BM-MSCs will release pro-inflammatory mediators into the conditioned medium or not.
In order to study CS-induced oxidative stress and inflammation in a short time frame, anacute (5-day) CS-exposed rat model was established in juvenile and adult groups. An age-dependent alteration of CS-induced oxidative and inflammatory responses was demonstrated in this model.
In summary, our in vivo rat model provides a platform for elucidating the effects of stem cell treatment in CS-induced oxidative stress and inflammation, leading to lung damage. Our findings suggest that treatment of IPSC-MSC or BM-MSC might be able to slow down CS-induced disease progression, possibly through anti-oxidant, anti-inflammatory and anti-apoptotic properties. However, caution should be taken as our in vitro data revealed that conditioned medium from MSCs may provoke pro-inflammatory responses. Further studies on the regulation of the activity of MSCs in vivo will be needed before developing IPSC-MSC into cell therapies for COPD to halt the progression over time.published_or_final_versionMedicineMasterMaster of Philosoph
Challenges to arterial endothelial function
No full textpublished_or_final_versionPharmacology and PharmacyDoctoralDoctor of Philosoph
Effects of Chinese green tea on cigarette smoke-induced oxidative stress, inflammation and proteases/anti-proteases in rat lung in vivo
No full textThe Best PhD Thesis in the Faculties of Architecture, Arts, Business &Economics, Education, Law and Social Sciences (University of HongKong), Li Ka Shing Prize, 2008-2009published_or_final_versionMedicineDoctoralDoctor of Philosoph
The involvement of serotoninergic system in cigarette smoke-induced oxidative stress and inflammation: relevantto chronic obstructive pulmonary disease
No full textCigarette smoking is a major risk factor in the development of age-related
chronic obstructive pulmonary disease (COPD) with chronic airway inflammation
as a key feature. Currently, no effective treatment can reduce the protracted
inflammation in the lung of COPD. Further research on the inflammatory
mechanisms would therefore be important in determining new potential
therapeutic targets in COPD. Serotonin (5-hydroxytryptamine, 5-HT) is a
neurotransmitter that plays an important role in pulmonary functions and
inflammatory responses. The serotoninergic system including serotonin
transporter (SERT), serotonin receptors (5-HTR) and its metabolic enzyme
monoamine oxidase (MAO) have been reported to associate with cigarette
smoking and/or COPD. Blockade of serotonin receptor 2A (5-HTR2A) with its
selective antagonist ketanserin has been shown to improve lung function in COPD
patients. In this study, we hypothesize that the serotoninergic system is involved
in cigarette smoke-induced oxidative stress, inflammation and COPD.
Exposure to cigarette smoke medium (CSM) caused the elevation of
interleukin (IL)-8 levels in primary normal human bronchial epithelial (NHBE)
cells and a human bronchial epithelial cell line (BEAS-2B) in vitro via activation
of p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling
pathway. Besides, CSM was found to disrupt the glutathione (GSH) system,
resulting in the translocation of nuclear factor-erythroid 2 related factor 2 (Nrf2)
to the nucleus. Knock-down of Nrf2 by small interference RNA (siRNA) blocked
CSM-induced IL-8 release. Pretreatment with ketanserin was found to attenuate
CSM-induced IL-8 release by inhibiting the p38, ERK1/2, and Nrf2 signaling
pathways, and by partially restoring the GSH system. On the other hand, CSM
reduced MAO activity in BEAS-2B, indicating a reduced catabolism of 5-HT.
Furthermore, 5-HT was found to share the common p38 and ERK1/2 signaling
pathway with CSM in IL-8 release.
In the cigarette smoke-exposed rat model, the GSH system in the lung was
found to be disrupted compared to the sham-air control, supporting our in vitro
findings. Interestingly, we found an increased MAO-A activity in the lung of
cigarette smoke-exposed rats in comparison to sham air-exposed rats. The
increased MAO-A activity in the lung was associated with the reduction of 5-HT
levels in bronchoalveolar lavage (BAL) and lung homogenates, while the
increased metabolism of 5-HT may be involved in cigarette smoke-induced
superoxide anion levels. On the other hand, serum, but not plasma level of 5-HT
was elevated in cigarette smoke-exposed group, which may be due to platelet
activation caused by cigarette smoke.
In the clinical study, the elevated plasma 5-HT levels were found to be
associated with an increased odds ratio for COPD and positively correlated with
age in COPD patients. Furthermore, plasma 5-HT was also demonstrated to be a
significant mediator on the relation between cigarette smoking and COPD.
In summary, our study supports the hypothesis that the serotoninergic
system contributes to cigarette smoke-induced oxidative stress, inflammation and
COPD. The serotoninergic system (e.g. 5-HTR2A) may constitute potential
therapeutic targets for the treatment of COPD, which is worthy for further
investigation.published_or_final_versionMedicineDoctoralDoctor of Philosoph
Development and optimization of KL4 peptide for pulmonary delivery of RNA
No full textInhalation of RNA therapeutics such as siRNA and mRNA has gained considerable attention for the treatment of a wide range of respiratory diseases. The delivery of RNAs presents a major obstacle that hinders their further development. A safe and effective vector is needed to mediate robust RNA transfection in the lung. In this study, KL4 peptide and its PEGylated derivatives were investigated for pulmonary delivery of RNA. KL4 is a synthetic cationic peptide with 21 residues containing repeating KLLLL subunits. It is structurally and functionally similar to surfactant protein B (SP-B) in human pulmonary surfactant. The rationale of employing KL4 as RNA delivery vector originates from the capability of naked RNA to transfect in the lung following pulmonary delivery. This observation leads to a hypothesis that cationic endogenous components in the lung may serve as natural RNA carriers by forming complexes with the anionic RNA, thereby promoting cellular uptake. The cationic SP-B is one of the possible candidates. Instead of investigating SP-B which is structurally complex, KL4 peptide was studied as RNA vector for pulmonary delivery.
The potential of KL4 to deliver siRNA was firstly evaluated. KL4 was able to bind to siRNA and form nano-sized complexes through electrostatic interaction. KL4/siRNA complexes showed superior stability in pulmonary surfactant and they mediated effective siRNA transfection in vitro on human lung epithelial cells. There was no significant sign of inflammatory response and toxicity in vitro at the tested dose. However, the high leucine content of KL4 renders it insoluble in water and limits its application as delivery vector. To address this problem, PEGylation strategy was applied by covalently attaching monodispersed polyethylene glycol (PEG) to KL4. Three PEGs with length varied between six to 24 monomers were employed to investigate the impact of PEGylation. The increase of PEG chain length resulted in higher water solubility, improved colloidal stability and enhanced transfection efficiency in vitro. Among all the peptides, PEG12KL4 peptide, which contains 12 monomers of PEG, was identified to be optimal for siRNA delivery as it attains a balance between good solubility, efficient transfection and high safety profile.
In addition to siRNA, the potential PEG12KL4 peptide for pulmonary delivery of mRNA was further investigated. PEG12KL4 peptide can bind and form complexes with mRNA. It mediated efficient mRNA transfection both in vitro in lung epithelial cells and dendritic cells as well as in the lungs of mice following intratracheal administration. Inhalable dry powder formulations of PEG12KL4/mRNA complexes were produced using spray drying (SD) and spray freeze drying (SFD) technologies. The dry powders exhibited satisfactory stability and aerosol properties for inhalation. More importantly, the transfection efficiency of PEG12KL4/mRNA complexes was well-preserved after drying.
To conclude, KL4 and PEGylated KL4 peptides are promising candidates for delivering RNA therapeutics in the clinic. Future studies will focus on applying the delivery system to disease models to evaluate their therapeutic potential.published_or_final_versionPharmacology and PharmacyDoctoralDoctor of Philosoph
The role of orosomucoid 1-like protein 3 (ORMDL3) in cigarette smoke-induced airway injury
No full textChronic obstructive pulmonary disease (COPD), as the third leading cause of mortality globally, is characterized by progressively persistent airflow limitation and chronic airway inflammation. Cigarette smoke (CS) is the major risk factor for COPD. Currently, no effective medications could reverse long-term decline in lung function of patients with COPD.
Orosomucoid 1-like protein 3 (ORMDL3), an endoplasmic reticulum (ER) transmembrane protein, is genetically associated with COPD, in addition to childhood-onset asthma. However, the impact of ORMDL3 in CS-induced injury of airway structural cells, including airway epithelial cells and airway smooth muscle cells, is still limited. In vivo CS-exposed animal study suggested that ORMDL3 was a CS-inducible protein with wide expression in rat lungs, including alveolar macrophages, airway epithelium and lesser extent in airway smooth muscle.
To investigate the role of ORMDL3 on ER stress in specific airway cell types in CS-induced airway injury, silencing ORMDL3 by using siRNA was performed in human bronchial epithelial cells (HBECs) and human airway smooth muscle cells (HASMCs) respectively. Differential regulations of ORMDL3 in CS-induced ER stress via unfolded protein response (UPR) pathways activation were found between HBECs and HASMCs in this study. In HBECs, ORMDL3 regulated cigarette smoke medium (CSM)-induced ER stress via inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) of UPR signalling pathways. ORMDL3 mediated CSM-induced activation of IRE1α, resulting in the generation of spliced X-box binding protein (XBP-1s) that indirectly induced inflammation via nuclear factor-κB (NF-κB) signalling pathway to activate the expression of inflammatory cytokine genes. On the other hand, the activation of ATF6 regulated downstream target sarco-endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) and subsequent protein folding and ER-associated degradation (ERAD) process. Furthermore, ATF6 caused activation of NF-κB signalling pathway through phosphorylation of Akt leading to airway inflammation and mucus hypersecretion. However, the study in HASMCs suggested that ORMDL3 regulated CSM-induced ER stress via protein kinase RNA-like ER kinase (PERK) and ATF6 arms of UPR signalling pathways. Activation of PERK induced transcription of CCAAT/enhancer-binding protein-homologous protein (CHOP) leading to airway smooth muscle proliferation. PERK activation also caused activation of NF-κB signalling pathway leading to airway inflammation. Similar to HBECs, ORMDL3 also regulated CSM-induced ER stress via activation of UPR-ATF6 pathway in HASMCs.
I have also established an effective three-dimensional co-culture model of well-differentiated HBECs and HASMCs to study the direct interaction. The findings showed greater responses in CSM-induced airway inflammation and mucus hypersecretion in co-culture model via regulation of ORMDL3-meditaed UPR-ATF6 pathway.
In summary, this study demonstrated for the first insight on the role of ORMDL3 in CS-induced airway injury, including airway inflammation, cell proliferation, mucus hypersecretion, ER stress, and mitochondrial dysfunction in HBECs and HASMCs respectively as well as in the novel co-culture model. The present findings provide evidence to suggest that ORMDL3 might be a novel therapeutic target for CS-mediated disease such as COPD.published_or_final_versionMedicineDoctoralDoctor of Philosoph
The role and mechanisms of interleukin-9 in facilitating anticancer effect of immune checkpoint blockade in lung cancer animal model
No full textLung cancer remains the major reason for cancer-related deaths. Immune checkpoint blockade (ICB), particularly with the use of programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) antibodies, has gained major advancement in cancer therapy throughout the recent decade, nonetheless only a minority of patients may benefit. Therefore, novel immunotherapeutic targets and combination strategies are urgently needed to
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expand the clinical benefit to wider patient population. Interleukin-9 (IL-9) is known to be important in modifying tumor biology and has pleiotropic effects in different cancers. In lung cancer, the function of IL-9 is not fully elucidated. The purpose of this study was to better understand the function(s) of IL-9 in lung cancer and the underlying mechanisms.
Lewis lung carcinoma (LLC) and CMT167 murine lung cancer cell lines were utilized in this study. IL-9 receptor (IL-9R) was only expressed in CMT167 cells, not LLC cells, but neither of their viability was altered by IL-9 in vitro. However, IL-9 led to tumor suppression and facilitated intratumoral T lymphocyte immunity in IL-9R-positive CMT167 model, not in IL-9R-lacking LLC model. Upon IL-9R knockdown, IL-9 lost its efficacy to induce anticancer immunity and tumor inhibition in CMT167 model.
In CMT167 tumors, although IL-9 elevated both tumor-infiltrating CD4+ and CD8+ T lymphocytes, it was the cytotoxic T cell subset that mediated the tumor suppression by IL-9. To understand the mechanisms of IL-9-enhanced CD8+ T cell responses in CMT167 tumors, T cell activation markers, recruitment chemokines, dendritic cells (DCs) frequencies, and tumor cell-surface major histocompatibility complex (MHC)-I expression were investigated. Increased DCs and upregulated MHC-I were observed in CMT167 tumors upon IL-9 therapy, both of which were
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absent in IL-9R knockdown CMT167 model. In vitro, IL-9 increased MHC-I expression in CMT167 and human lung adenocarcinoma A549 cells consistently. The signal transducer and activator of transcription 1 (STAT1) and protein kinase B (Akt) pathways in CMT167 cells were not affected, while phosphorylation of extracellular signal-regulated kinase (ERK) was enhanced by IL-9. The upregulation of MHC-I by IL-9 in CMT167 cells was abrogated by ERK inhibition.
Previously, others have reported a positive correlation of IL-9-producing T helper type 9 (Th9) cells with the therapeutic efficacy of PD-1 blockade in cancers, demonstrating that IL-9 and PD-1/PD-L1 inhibition might have a potential synergism. Indeed, besides the enhanced anti-tumor T cell immunity, IL-9 also increased the PD-1 expression on CD8+ T lymphocytes and PD-L1 expression on CMT167 cells. Combined treatment with IL-9 and PD-1 blockade further increased intratumoral cytotoxic T lymphocytes and synergistically inhibited the growth of CMT167 tumors.
In summary, IL-9/IL-9R interaction in CMT167 tumors could promote tumoral MHC-I presentation and increase DCs frequencies, leading to tumor growth suppression by enhanced cytotoxic T lymphocyte immunity. Intratumoral IL-9R expression might potentially be investigated as a selection biomarker for lung cancer subsets susceptible to IL-9 therapy. The findings from this study lend support
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for future clinical evaluation of IL-9 as an immunomodulatory agent in combination with PD-1 blockade in lung cancer.published_or_final_versionMedicineDoctoralDoctor of Philosoph
Therapeutic arginine depletion in small cell lung cancer : preclinical activity, mechanisms and resistance
No full textSmall cell lung cancer (SCLC) is an aggressive malignancy, which is characterized by rapid doubling time, good initial response to chemoradiotherapy, but early recurrence with distant metastasis, resulting in extremely poor overall prognosis. Current chemotherapeutic regimens in SCLC are still the time-honored etoposide/cisplatin in the first-line and topotecan in the second-line, which lack specificity with serious adverse events. Therefore, there is an urgent need to explore novel drugs to improve the clinical outcomes of SCLC.
Arginine is a non-essential amino acid to human, but certain tumor cells cannot synthesize arginine endogenously. Arginine depletion has become a potential targeted therapy for auxotrophic tumors including human hepatocellular carcinoma, melanoma, leukemia and mesothelioma. BCT-100, a pegylated recombinant human arginase, can catalyze arginine to ornithine, leading to arginine depletion in tumor cells. Our aim is to investigate the preclinical activity and mechanism of BCT-100 in SCLC in vitro and in vivo. In addition, the BCT-100 acquired resistant cell lines derived from SCLC were employed to explore the underlying resistance mechanism to BCT-100 in SCLC.
In the first part, most (7/9) of the SCLC cell lines tested were sensitive to BCT-100 treatment. H446, H510A and H526 were relatively sensitive to BCT-100 treatment and chosen for further study on the underlying mechanisms in vitro. BCT-100 induced cytotoxicity via arginine depletion accompanying with oxidative stress and cell cycle arrest in sensitive cell lines. Hydrogen peroxide (H2O2) and superoxide (O2-) were increased and glutathione (GSH) was decreased, accounting for oxidative stress triggered by BCT-100. Co-treatment with N-acetyl-L-cysteine (NAC) reversed the cytotoxic effect mediated by ROS generation, while buthionine sulfoximine (BSO) enhanced the apoptosis induced by BCT-100 treatment. Besides, mitochondrial membrane depolarization (MMD) led to cytochrome c and smac releasing from mitochondria to cytoplasm, suggesting mitochondrial dependent apoptosis. The G1 cell cycle arrest related biomarkers cyclin A2, cyclin B1, CDK2 and CDK4 were down-regulated in a time-dependent manner upon BCT-100 treatment. AKT-mTOR signaling pathway was activated and co-incubation with mTOR inhibitor rapamycin potentiated the cytotoxic effect of BCT-100.
In the second part, H446 and H510A cells were employed to establish xenograft models in mice. BCT-100 suppressed the tumor growth and prolonged the median survival time significantly in a dose-dependent manner in both xenografts, confirming the in vitro activity observed in earlier part of study. Apoptosis and cell cycle arrest were observed to explain antitumor effect of BCT-100 in vivo.
In the third part, BCT-100 acquired resistant cells (H446-BR and H526-BR) were obtained after long-term exposure. Compared with parental cells, resistant cells exhibited more aggressive migration features, anoikis resistance and epithelial-mesenchymal transition (EMT) phenotype. Contactin-1 (CNTN-1) was fished out by gene chip assay as it was up-regulated in resistant cells, and silencing CNTN-1 re-sensitized H446-BR and H526-BR to BCT-100 treatment. Besides, knockdown of CNTN-1 attenuated the EMT progression through inhibiting AKT signaling pathway.
In summary, BCT-100 was shown to have anticancer effect in SCLC via arginine depletion, oxidative stress, cell cycle arrest and apoptosis. CNTN-1 enhanced the acquired drug resistance through induction of EMT progression by AKT signaling pathway.published_or_final_versionMedicineDoctoralDoctor of Philosoph
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