Thromboxane A2 (TXA2) and ET-1 have been known to play important roles in modulating vascular contraction and growth. The present study was undertaken to examine the effect of TXA2 on the induction of endothelin-1 (ET-1) mRNA and protein levels in smooth muscle cells derived from rat heart. U-46619, a stable TXA2 mimetic, superinduced preproET-1 mRNA in the presence of cycloheximide in these cells. This effect could be blocked by SQ-29548, a TXA2/prostaglandin H2 receptor antagonist and by actinomycin D, an RNA synthesis inhibitor. In addition, H7, a protein kinase C inhibitor, could abolish the induction. Transient transfection experiment revealed that the elevated ET-1 mRNA level after U-46619 treatment was a result of the activation of ET-1 gene activity. The elevated ET-1 message level was accompanied by increased ET-1 release into the cultured medium. These results show that the short-lived TXA2 can induce potent and long-lived ET-1. These findings support a potential role for ET-1 in the pathogenesis of coronary atherosclerosis and hypertension evoked by TXA2.

No / Previous studies in this laboratory have suggested that the isolated uterus from non-pregnant mice has a prostaglandin F and a thromboxane receptor population similar to that found in human myometrium. The aim of this study was to investigate any regional variation in myogenic activity ) and the and responsiveness to prostaglandin F(2alpha) (PGF(2alpha) thromboxane mimetic U46619 in the mouse uterus taken during different stages of the oestrous cycle and during pregnancy. Uterine samples from BKW mice were taken from different anatomical segments along the length of each uterine horn and set up for superfusion at 2 ml/min with Krebs solution (containing 1 microM indometacin) at 37 degrees C, and gassed with 95%O(2)/5%CO(2). Responses (area under the curve) are expressed as a percentage of the final contraction induced by hypotonic shock. Data are expressed as the means +/- s.e.m. of n=5-12 and were analysed using Student's paired t-test or two-way ANOVA with a Bonferroni post hoc test. Regional variation in myogenic activity was observed in all tissues studied except those taken during labour. These tissues displayed significantly greater myogenic activity than tissues taken at late gestation and at all stages of the oestrous cycle. Tissues from pregnant animals were generally more responsive to U46619 and PGF(2alpha) than tissues taken from non-pregnant animals. Tissues taken from the upper segment of the uterine horn were more responsive to both agonists during the oestrous cycle. The findings demonstrated that the hormonal milieu and site of excision are important for myogenic activity and responsiveness.

Cardiovascular diseases are a major cause of mortality and morbidity in the developed countries. Anti-platelet therapy is a cornerstone treatment for patients with cardiovascular diseases. Patients are routinely managed with a combination therapy consisting of aspirin and clopidogrel. Aspirin inhibits cyclooxygenase 1 (COX 1) a crucial intermediate enzyme involved in thromboxane biosynthesis. Clopidogrel on the other hand antagonizes ADP receptor P2Y12. ADP is a weak platelet agonist stored in platelet dense granules and is released upon platelet activation. ADP activates platelets through two purinergic receptors namely P2Y1 and P2Y12 these receptors couple to Gq and Gi class of G-proteins, respectively. P2Y1 causes calcium mobilization through activation of PLC-β. P2Y12 inhibits adenylyl cyclase, causes activation of Rap1B and Akt. Signaling from both the receptors is required for complete integrin activation, thromboxane generation and Erk activation. Previous studies have shown that P2Y12 potentiates fibrinogen receptor activation, secretion, thrombi stabilization, thrombin generation, platelet leukocyte aggregation formation. ThromboxaneA2 (TXA2) is a potent platelet agonist generated through arachidonic acid metabolism in platelets. TXA2 thus, generated after platelet activation acts as a positive feedback mediator along with ADP. Under physiological conditions, platelet activation leads to thrombin generation through coagulation cascades. Generated thrombin activates PAR receptors and ADP is released from dense granules, which further potentiates thromboxane generation downstream of PARs. Current anti-platelet therapy regimens often include P2Y12 antagonists and aspirin in management of patients with acute coronary syndrome (ACS) and in those undergoing percutaneous coronary intervention (PCI) with stent implantation. However, there still exists a need for improved treatment strategies as not all patients benefit from this dual combination therapy. Reasons include, poor responders either to P2Y12 antagonists or to aspirin, or if aspirin is contraindicated in these patient populations. In the current study we evaluated the role of P2Y12 in thromboxane generation under physiological conditions. We studied serum thromboxane generation in a model system wherein P2Y12 was antagonized or deficient. Using pharmacological approaches we show that dosing mice with 30mg/Kg/body weight clopidogrel or 3mg/Kg/body weight prasugrel decreased serum thromboxane levels when compared to the control mice. Pre-treatment of human blood ex vivo with active metabolites of clopidogrel (R361015) or prasugrel (R138727) also led to reduction in thromboxane levels. We also evaluated serum thromboxane levels in P2Y receptor null mice, serum thromboxane levels in P2Y1 null mice were similar to those in wild type littermates, and were inhibited in P2Y12 null mice. Furthermore, serum thromboxane levels in P2Y12 deficient patients, previously described in France and Japan, were also evaluated and these patients had lower serum thromboxane levels compared to normal controls. In a pilot study, serum thromboxane levels were radically reduced in healthy human volunteers upon dosing with clopidogrel, compared to the levels before dosing. In conclusion, P2Y12 antagonism alone can decrease physiological thromboxane levels. Thus P2Y12 regulates physiological thromboxane levels. Further it is known that ADP-induced thromboxane generation is integrin-dependent. However it is not clear if other potent platelet agonists like thrombin require outside-in signaling for thromboxane generation. Our results show that thrombin-induced thromboxane generation was independent of integrins i.e. when platelets were stimulated with PAR agonists in presence of fibrinogen receptor antagonist thromboxane generation was not affected. Since PAR agonists, unlike ADP, activate G12/13 signaling pathways. Hence, we hypothesized that these pathways might play a role in TXA2 generation. Our results show, that inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by costimulation of G12/13 pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G12/13 pathways. Next, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. Our results showed that c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. We observed differential activation of FAK downstream of integrins and G12/13 pathways. ADP-induced activation of FAK was integrindependent and SFK-independent. On the other hand selective activation of G12/13 pathway lead to FAK activation, in SFK and Rho dependent manner. We also evaluated specificity of new FAK inhibitor TAE-226 to understand the role of FAK in TXA2 generation. Our results showed that TAE-226 exhibited non-specific effects at higher concentrations. Furthermore, in comparison to WT mice, FAK null mice did not show any difference in TXA2 generation. Therefore, we concluded that differential activation of FAK occurs downstream of Integrins and G12/13 pathways. However, the common effector molecule downstream of integrins and G12/ 13 pathways contributing to TXA2 generation in platelets remains elusive. / Molecular and Cellular Physiology

Platelets are primary components of hemostasis. However, incongruous activation of platelets lead to thrombosis, which result in multiple cardio-vascular and cerebrovascular complications. Thus, platelet activation is tightly regulated. Molecular components that aid in activation of platelets have been extensively studied. However, molecular pathways that negatively regulate platelet activation and prevent accidental activation of platelets are poorly understood. In this study we investigated the molecular mechanisms that negatively regulate platelet activation. Protein Kinase C isforms (PKCs) are serine threonine kinases that regulate various platelet functional responses leading to hemostasis. Positive regulatory role of PKCs towards platelet aggregation and secretion has been extensively studied. However, we have recently demonstrated that PKCs negatively regulate ADP- induced thromboxane generation. The PKC isoforms and mechanism involved in this process have not been known. Thus, in this study we investigated the mechanism by which PKCs negatively regulate ADP-induced thromboxane generation and identified PKC isoforms that regulate thromboxane generation. Thromboxane generation in platelets is a multi-step process beginning with cPLA2 activation. cPLA2 activation is the rate limiting step in the process of thromboxane generation. Furthermore, cPLA2 activation is regulated by ERK and calcium in various cell systems including platelets. PKC inhibition potentiated both cPLA2 and ERK activation, suggesting that PKCs negatively regulate thromboxane generation by regulating ERK activation, which in turn regulates cPLA2 activation. Furthermore, we have also shown that PKCs negatively regulate ADP-induced calcium mobilization. ADP activates platelets via P2Y1 and P2Y12 receptors. P2Y12 receptor-mediated signaling is shown to positively regulate P2Y1-mediated calcium mobilization in platelets. Furthermore, PKCs are shown to negatively regulate P2Y12 receptor desensitization in platelets. Thus, we investigated if PKCs regulate calcium mobilization indirectly by regulating P2Y12 receptor function. However, PKCs regulate calcium mobilization independent of P2Y12 receptor signaling. In summary we have shown that PKC isoforms negatively regulate ADP-induced thromboxane generation by regulating calcium mobilization and ERK activation that in turn regulates cPLA2 activity. We further investigated the PKC isoforms involved in this process. Based on our results with Go-6976, a classical PKC inhibitor and GF109203X, a pan PKC inhibitor, we identified that that novel or atypical PKC isoforms are involved in negative regulation of ADP-induced thromboxane generation. Thus, we investigated the role of various novel class of PKC isoforms (nPKCs) in platelets. We first investigated the nPKCs activated by ADP. In aspirin-treated platelets, ADP failed to activate nPKC θ and δ non-stirring conditions. Thus, we conclude that these isoforms are not involved in negative regulation of thromboxane generation. We further investigated if other non-classical PKC isoforms i. e nPKC η and ε or atypical PKC isoforms could be involved in this process. We began our investigation with the mechanism of activation and functional role of nPKC η in platelets. The mechanism of activation of PKCs has been extensively studied in various cell systems including platelets. However, the mechanism by which they are inactivated is not completely understood. In this study, we demonstrate a novel mechanism of inactivation of nPKC η isoform by integrin associated serine/threonine phosphatase. we demonstrated that ADP activates nPKC η via P2Y1 receptor coupled to Gq. As expected, Gi pathway, which does not generate DAG or mobilize calcium, has no role in regulation of nPKC η. Interestingly, we show that upon activation of platelets, αIIbβ3 mediated outside-in signaling dephosphorylates nPKCη through PP1γ phosphatase. We have also evaluated the role of nPKC η using η-RACK antagonistic peptides that interfere with enzyme-substrate interaction. Similar antagonistic peptides have been successfully used in various cell systems such as cardiomyocytes and neuronal cell. Using η-RACK antagonists we have demonstrated that nPKC η positively regulates agonist- induced thromboxane generation with no effect on agonist- induced platelet aggregation. The peptides were targeted in to the cell using TAT carrier protein, which is also used as a negative control for these experiments. The specificity of η-RACK antagonistic peptides is further elucidated by the fact that they do not affect the platelet aggregation. In summary, nPKC η is activated by ADP via P2Y1 receptor and is dephosphorylated by integrin αIIbβ3 via PP1γ phosphatase. Furthermore, activated nPKC η positively regulates ADP- induced thromboxane generation with no effect on aggregation. Since, our aim was to investigate the nPKC isoforms that negatively regulate ADPinduced thromboxane generation we investigated if nPKC ε is involved in this process. We made use of PKC ε knockout mice (PKC ε KO) for this process. We observed potentiated thromboxane generation in ADP-induced PKC ε murine platelets compared to witd type murine platelets. Thus, PKC ε might be one of the PKC isoforms involved in negative regulation of ADP-induced thromboxane generation. However, we failed to detect PKC ε in human platelets using western blot analysis. Since, PKC ε has been reported to be a part of platelet kinase repertoire, it could be limitation of our technique that we failed to detect it in western blot analysis. Since, PKCs negatively regulate ADP-induced thromboxane generation, we also investigated if PKCs also regulate PAR-mediated thromboxane generation. Similar to ADP, PAR-mediated thromboxane generation is not affected by Classical PKC isoforms. Furthermore, although non-classical PKC isoforms negatively regulate thromboxane generation, the extent of negative regulation is smaller and non-significant compared to ADP. Thus, we investigated if activation of nPKC isoforms were different between ADP and AYPGKF (PAR4 agonist). While, ADP fails to activate nPKC δ and θ, PARs activate Them. Furthermore, we have recently demonstrated that nPKC δ and θ are positive regulators of PAR-mediated platelet functional responses. Therefore, PKCinduced potentiation of thromboxane generation by ADP and PAR agonist are different due to differential activation of PKCs. This data lead to our final project, where we investigated the reason for differential activation of nPKC isoforms by various platelet agonists. Using strong and weak platelet agonists and DAG analogue, DiC8, we demonstrated that different platelet agonists differentially regulate nPKC activation due to variable amounts of DAG generated by them. Furthermore, we also have demonstrated that nPKC η and ε have higher affinities to DAG compared to nPKC δ and θ. / Molecular and Cellular Physiology

The pathogenesis of inflammatory liver diseases and development of liver fibrosis involves hepatocytes as well as non-parenchymal liver cells like resident liver macrophages (Kupffer cells (KC)), Stellate cells and endothelial cells. Kupffer cells play a critical role in liver (patho)physiology and in the defense of the liver during inflammation. They constitute about 50% of non-parenchymal cells and are the largest population of tissues macrophages in the body. Infections, toxins (lipopolysacharide (LPS)), parenchymal damage and stresses stimulate the inflammatory response of Kupffer cells with the following secretion of bioactive factors, cytotoxicity, antigen processing, etc. Resident liver macrophages are the main producers of inflammatory mediators in the liver. Among them there are prostanoids (prostaglandin (PG) E2 and thromboxane (Tx) A2), cytokines (e.g. interleukin (IL)-1,-6, -10, tumor necrosis factor (TNF) α) and inorganic mediators like nitric oxide (NO). Macrophages-derived products play opposing roles in the development of liver fibrogenesis: IL-1β, TNFα, IL-6, transforming growth factor (TGF)-β and TxA2 (pro-fibrogenic mediators) promote whereas PGE2, IL-10 and nitric oxide (anti-fibrogenic mediators) suppress liver fibrogenesis. The present study shows the production of PGE2 and TxA2 by resident liver macrophages upon prolonged activation by LPS and the characterization of biosynthesis pathways. The production of PGE2 and TxA2 is followed during 24 h after stimulation of macrophages with LPS. The involvement of enzymes is measured on the RNA level (RT-PCR), protein level (Western blot analysis) and activity (activity assays), respectively. The amounts of released prostanoids are measured at time points 2, 4, 8 and 24 h after LPS stimulation. The production of PGE2 is very low without stimulation, shows a delay within the first few hours after stimulation with LPS, and thereafter linearly increases up to 24 h. TxA2 production is very low without stimulation, and increases without a time-delay after the addition of LPS. Prostanoid biosynthesis is inhibited by dexamethasone. The present study shows the involvement and regulation of the AA cascade by the following enzymes: cPLA2: is expressed in resting Kupffer cells; cPLA2 expression and phosphorylation is increased by LPS, dexamethasone suppresses the LPS effect, localization in membrane fraction. COX-1: is expressed in resting Kupffer cells; COX-1 expression is not influenced by LPS and dexamethasone. The COX-1 inhibitor SC560 suppresses the LPS-induced production of PGE2 and TxA2 (8h and 24h), localization predominantly in membrane fraction. COX-2: is almost not expressed in resting Kupffer cells; COX-2 expression is highly increased by LPS, dexamethasone suppresses the LPS effect. The COX-2 inhibitor SC236 inhibits the production of PGE2 and TxA2 at 8h by about 77% and 20%, and at 24h by about 42% and 34%, respectively, localization predominantly in membrane fraction. mPGES-1: is almost not expressed in resting cells; mPGES-1 expression is highly increased by LPS, dexamethasone suppresses the LPS effect, localization in membrane fraction. mPGES-2: is expressed in resting Kupffer cells; mPGES-2 expression is slightly increased by LPS, localization predominantly in membrane fraction. cPGES: is expressed in resting Kupffer cells; LPS has no effect, localization predominantly in soluble fraction. TxA2 synthase: is expressed in resting Kupffer cells; LPS and dexamethasone have no effect, localization predominantly in membrane fraction. Treatment of Kupffer cells with IL-1ß and TNF-α leads to an enhanced release of PGE2 and TxA2 and upregulate the expression of cPLA2, COX-2 and mPGES-1. IL-6 has no effect on prostanoid production. In contrast, IL-10 suppresses the LPS-induced production of PGE2 and TxA2 and expression of cPLA2, COX-2 and mPGES-1. Resting Kupffer cells release very low amounts of NO and do not express iNOS, nNOS and eNOS. LPS, TNF-α and IL-1ß upregulate NO release and the expression of iNOS whereas dexamethasone and IL-10 downregulate NO release and the expression of iNOS. PGE2 suppresses the LPS-induced release of NO but enhances the cytokine-induced release of NO. NO induces a release of PGE2. Thus, the study demonstrates a crosstalk between prostanoids, nitric oxide and cytokines in Kupffer cells under inflammatory conditions and demonstrates a possible anti-fibrogenic effect of PGE2 in the process of liver fibrogenesis.

肺癌是一個世界性的健康難題。大量研究證據顯示，煙草及其致癌物NNK對環氧酶（COX）-2及其下游產物具有促進效應。血栓素（TxA）2是COX-2的關鍵性下游產物之一，該論文闡述了TxA2在NNK導致的肺癌增長中的可能作用。 / 我們發現相对于非吸烟者，吸煙者肺癌組織表达更高水平的TxA2合酶（TxAS）。NNK可以刺激培養的肺癌細胞TxA2合成。用TxAS抑制劑和TxA2受體（TP）拮抗劑分別阻抑TxA2的合成與功能可以引起細胞凋亡，從而有效抑制NNK導致的細胞增殖效應。在TxA2合成受抑制的情況下，TP激動劑U46619幾乎可以重建NNK效應，說明TP在NNK效應中的重要作用。研究還顯示，激活的TP可以通過PI3K/Akt和ERK通路進一步激活CREB，從而參與NNK對肺癌細胞的促生長效應。 / 緊接著，我們的研究顯示TP 可以調節NNK對COX-2 和TxA2的誘導，而且發現NNK刺激的TxA2合成主要依賴於COX-2活性。COX-2和TxA2功能抑製劑對NNK的促細胞生長作用具有相似的抑制效用。考慮到TP是TxA2的功能受體，該資料說明TP在NNK處理的肺癌細胞中傳遞了上游因子COX-2的促腫瘤作用。在使用COX-2小干擾RNA（siRNA）抑制NNK作用的情況下，TP激動劑U46619幾乎可以恢復NNK的效應證實了TP的傳遞者角色。研究還發現 TPα而不是TPβ在培養的肺癌細胞系中廣泛表達，並且過表達TPα具有促進腫瘤生長的作用。在用NNK處理細胞的條件下，TPα還具有促COX-2表達和TxA2生成的作用。 / 我們的研究進一步發現，在吸煙者肺癌組織中TPα表達增高，這與TxAS的表達相似。与此结果相一致，在經NNK處理的A/J小鼠肺癌組織中，TxAS和TP表達水準也是明顯上升的。在細胞培養實驗中，NNK能夠提高TxAS蛋白和信使RNA（mRNA）的表達水準。但是，在TP的兩個亞型TPα和TPβ中， NNK僅能促進TPα的蛋白表達，對它們的mRNA均無影響。NNK對TxAS的促表達作用是核轉錄因數(NF)-κB依賴性的。其他的幾個關鍵轉錄因數，諸如特異性蛋白(SP)-1，CREB和活化受體 （PPAR）γ均未參與NNK對TxAS和TPα的表達促進作用。進一步的，轉錄後機理被證實參與了NNK對TPα的作用。TPα而不是TPβ經鑒別在NNK的促NF-κB 激活 和 促TxAS 表達效應中起正向調節作用。 / 總之， 我們的研究說明TxA2相關通路在NNK的促肺癌細胞生長效應中起正向調節作用。我們的研究揭示了TPα的自我激活環路。通過該環路，TxA2，或者說TxAS和TPα參與了NNK的肺癌促生長效應。因此，我們的研究為肺癌的防治了提供了一個新的方向，即靶向TxAS和TPα是一種可能有效的策略。 / Lung cancer concerns a world-wide health problem. There is considerable evidence of that tobacco smoke and its carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) have the potential effects on the production of cyclooxygenase (COX)-2 and its downstream products in tumor cells. This thesis is constructed to describe the study focused on the role of thromboxane A2 (TxA2), one of the key downstream products of COX-2, in NNK-induced lung tumor growth. / We found that as compared to non-smokers, lung cancer tissues obtained from smokers tended to express more TxA2 synthase (TxAS). Moreover, NNK could stimulate TxA2 synthesis in lung cancer cells. Blockade of TxA2 synthesis and action by TxAS inhibitor and TxA2 receptor (TP) antagonist completely blocked NNK-promoted cell proliferation via inducing apoptosis. Moreover, TP agonist U46619 reconstituted a near full proliferative response to NNK when TxAS was inhibited, affirming the role of TP in NNK-induced cell growth. Furthermore, we revealed that the activated TP may then activate CREB through PI3K/Akt and ERK pathways, thereby contributing to the NNK-induced lung cancer cell growth. / We subsequently showed that TP could modulate the induction of COX-2 and TxA2 by NNK. The synthesis of TxA2 stimulated by NNK was found to be mainly dependent on COX-2 activity. Intriguingly, there are similar inhibitory effects on NNK-induced cell growth between pharmacological inhibition of COX-2 and the blockade of TxA2 synthesis and action. Because TP is the natural receptor of TxA2, these results suggest that TP may function as a mediator for the tumor-promoting effects of COX-2 upon NNK treatment, which was confirmed by the data showing that U46619 almost restored NNK effects in the presence of COX-2-siRNA. Importantly, TPα, but not TPβ was found to be widely expressed in lung cancer cells and be able to promote tumor growth, COX-2 expression and TxA2 synthesis upon NNK treatment. / We further demonstrated that in lung tumor tissues obtained from smoker, TPα protein was increased, which was similar to the change in TxAS protein. The increased levels of TxAS and TP proteins were also found in lung cancer tissues of A/J mice treated with NNK. In cell culture experiments, NNK could increase TxAS at both protein and mRNA levels. However, TPα rather than TPβ was increased by NNK at protein but not mRNA level. NNK-stimulated TxAS expression was dependent on nuclear factor (NF)-κB signaling. Other key transcriptional factors, such as specificity protein(SP)-1, CREB and peroxisome proliferator-activated receptor-gamma (PPARγ), were not involved in NNK-induced TxAS and TPα expression. Further experiments revealed that post-transcriptional mechanisms were responsible for NNK-induced TPα expression. TPα rather than TPβ was finally identified to have a positive role in NNK-induced NF-κB activation and TxAS expression. / Taken together, our study suggests that TxA2 pathway has a positive role in NNK-induced lung cancer cell growth. An auto-positive feedback loop of TPα activation to facilitate lung tumor growth in the presence of NNK is delineated by these results. Therefore, targeting TxAS or/and TPα may represent a promising strategy for prevention and treatment of lung cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Huang, Runyue. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 119-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.I / 摘要 / Publications / Acknowledgement / Abbreviations / Table of contents / Chapter Chapter 1 --- General introduction--Tobacco smoking, COX-2 pathway and cancer / Chapter 1.1 --- Abstract --- p.1 / Chapter 1.2 --- Introduction --- p.2 / Chapter 1.3 --- Cyclooxygenase and prostanoids --- p.5 / Chapter 1.4 --- The effects of tobacco smoking on COX-2 pathway, and the related pathologies --- p.8 / Chapter 1.4.1 --- Smoking, PGE2, inflammation and immunosupression --- p.8 / Chapter 1.4.2 --- Smoking, TxA2, platelet activation, cell contraction and angiogenesis --- p.11 / Chapter 1.4.3 --- Smoking and PGI2 --- p.16 / Chapter 1.5 --- The role of cyclooxygenase-2 pathway in the progression of tobacco smoke-related cancers --- p.19 / Chapter 1.5.1 --- Lung cancer --- p.19 / Chapter 1.5.2 --- Gastrointestinal cancer --- p.23 / Chapter 1.5.3 --- Bladder cancer --- p.24 / Chapter 1.5.4 --- Head and neck squamous cell carcinoma --- p.25 / Chapter 1.5.5 --- The signaling mechanisms underlying the induction of COX-2 by smoking in tumors --- p.26 / Chapter 1.6 --- Summary, future directions and key questions --- p.28 / Chapter Chapter 2 --- NNK induces lung cancer cell growth by stimulating TxA2 and its receptor / Chapter 2.1 --- Abstract --- p.32 / Chapter 2.2 --- Introduction --- p.33 / Chapter 2.3 --- Materials and Methods --- p.35 / Chapter 2.3.1 --- Cell lines and cell culture --- p.35 / Chapter 2.3.2 --- Chemicals and drug treatment --- p.35 / Chapter 2.3.3 --- Thromboxane B2 EIA assay --- p.36 / Chapter 2.3.4 --- MTT assay --- p.36 / Chapter 2.3.5 --- BrdU cell proliferation assay --- p.37 / Chapter 2.3.6 --- Flow cytometry for analysis of apoptosis --- p.37 / Chapter 2.3.7 --- Transfection of cells with CREB siRNA --- p.38 / Chapter 2.3.8 --- Western blot analysis and antibodies --- p.38 / Chapter 2.3.9 --- Statistical analysis --- p.39 / Chapter 2.4 --- Results --- p.41 / Chapter 2.4.1 --- High expression of TxAS in lung cancer tissues of smoker --- p.41 / Chapter 2.4.2 --- NNK stimulated TxA2 synthesis in lung cancer cells --- p.43 / Chapter 2.4.3 --- Blockade of TxA2 synthesis and action prevented NNK-induced cell growth --- p.44 / Chapter 2.4.4 --- TxA2 mimetic U46619 reconstituted NNK-enhanced cell proliferation under TxA2-inhibited condition --- p.47 / Chapter 2.4.5 --- Blockade of TxA2 synthesis or action induced the apoptosis of the NNK-exposed cells --- p.47 / Chapter 2.4.6 --- CREB is accountable for the key role of TxA2 in NNK-enhanced cell proliferation --- p.49 / Chapter 2.4.7 --- PI3K/Akt and ERK rather than JNK and p38 pathways were mediated by TxA2 in the NNK-exposed cells --- p.52 / Chapter 2.4.8 --- CREB is located downstream of the PI3K/Akt and ERK pathways in NNK-treated cells --- p.53 / Chapter 2.5 --- Discussion --- p.55 / Chapter Chapter 3 --- The positive role of TPα in the induction of COX-2, TxA2 and cell growth by NNK in human lung cancer cells / Chapter 3.1 --- Abstract --- p.62 / Chapter 3.2 --- Introduction --- p.63 / Chapter 3.3 --- Materials and methods --- p.65 / Chapter 3.3.1 --- Cell culture and chemicals --- p.65 / Chapter 3.3.2 --- Transient transfections --- p.66 / Chapter 3.3.3 --- TxB2 measurement --- p.66 / Chapter 3.3.4 --- Cell growth detection --- p.67 / Chapter 3.3.5 --- Analysis of apoptosis --- p.67 / Chapter 3.3.6 --- Western blot analysis and antibodies --- p.67 / Chapter 3.3.7 --- Statistical analysis --- p.68 / Chapter 3.4 --- Results --- p.70 / Chapter 3.4.1 --- Examination of TP as the modulator for induction of COX-2 and TxA2 by NNK --- p.70 / Chapter 3.4.2 --- The TxA2 generated in cells treated with NNK is mainly dependent on COX-2 activity --- p.72 / Chapter 3.4.3 --- Examination of TP as the key mediator for the tumor-promoting effect of COX-2 --- p.72 / Chapter 3.4.4 --- The expression and action of α and β isoforms of TP in human lung cancer cells --- p.77 / Chapter 3.4.5 --- the identification of positive role of TPα in NNK-induced COX-2, TxA2 and cell growth in lung cancer cells --- p.79 / Chapter 3.5 --- Discussion --- p.81 / Chapter Chapter 4 --- TP-α facilitates lung tumor growth through an autoregulatory feedback mechanism / Chapter 4.1 --- Abstract --- p.88 / Chapter 4.2 --- Introduction --- p.89 / Chapter 4.3 --- Materials and methods --- p.91 / Chapter 4.3.1 --- Human lung tissue and immunohistochemical analysis --- p.91 / Chapter 4.3.2 --- Animal treatment --- p.91 / Chapter 4.3.3 --- Cell culture and chemicals --- p.92 / Chapter 4.3.4 --- Transient transfection --- p.93 / Chapter 4.3.5 --- Real-time PCR --- p.93 / Chapter 4.3.6 --- Western blot analysis and antibodies --- p.94 / Chapter 4.3.7 --- Statistical analysis --- p.95 / Chapter 4.4 --- Results --- p.96 / Chapter 4.4.1 --- The effects of smoking on the expression of TP in human lung cancer tissue --- p.96 / Chapter 4.4.2 --- The effects of NNK on the expression of TxAS and TP in lung tissues of A/J mice --- p.98 / Chapter 4.4.3 --- The effects of NNK on the expression of TxAS and TPα in lung cancer cells --- p.99 / Chapter 4.4.4 --- Identification of the roles of NF-κB, CREB and SP1 in NNK-induced TxAS and TPα expression --- p.101 / Chapter 4.4.5 --- The negative role of PPARγ in NNK-induced TxAS and TPα expression --- p.104 / Chapter 4.4.6 --- NNK-induced TPα expression via post-transcriptional mechanism --- p.105 / Chapter 4.4.7 --- Examination of TPα auto-activation mechanism in lung cancer cells stimulated with NNK --- p.106 / Chapter 4.5 --- Discussion --- p.109 / Chapter Chapter 5 --- Conclusion and future works / Chapter 5.1 --- Conclusion --- p.114 / Chapter 5.2 --- Future works --- p.115 / Chapter 5.2.1 --- The possible role of miR-34c in the auto-regulatory loop of TxAS expression or TPα activation --- p.116 / Chapter 5.2.2 --- The possible role of FOXO3a in the auto-regulatory loop of TxAS expression or TPα activation --- p.116 / References --- p.119

Cell membrane receptors help to mediate communication between the cell and its environment. The largest group of these membrane receptors belong to the family of G protein-coupled receptors (GPCRs). GPCRs contain seven transmembrane (TM) helices and signal predominantly through heterotrimeric G proteins in response to diverse extracellular stimuli. Previously, three levels of amino acid conservation were proposed to understand the structure and function of a GPCR. This includes “signature” amino acids, “group –conserved” amino acids and amino acids conserved only within a specific subfamily. The group-conserved residues in class A GPCR family involve amino acid conservation of up to 99% when considered as a group of small and weakly polar residues (Ala, Gly, Ser, Cys and Thr). These group-conserved residues have been proposed as key determinants in helix-helix interactions. Therefore, I selected these residues for structure-function analysis in the amine and the prostanoid receptor sub-families of class A GPCRs. Molecular and biochemical assays clearly demonstrate the importance of group-conserved residues in β2-adrenergic receptor and thromboxane A2 receptor (TP) structure and function. These studies led to the identification of a non-synonymous single nucleotide polymorphic variant (nsSNP) A160T in TP to be a constitutively active mutant (CAM). Further, the TP-CAM was used as a pharmacological tool that enabled classification of well-known TP-blockers, into neutral antagonists and inverse agonists. The role of TP-A160T in prostanoid receptors, TP- Prostacyclin receptor (IP) heterodimerization and signaling was investigated. Activation of a GPCR ultimately leads to structural changes in its intracellular loops (ICLs), which in turn activates G-protein. TP activates its cognate G protein (Gαq), while IP mediates signaling, through Gαs. Using TP-IP chimeric receptors, molecular modelling, and site directed mutagenesis studies I determined the specific ICL regions required for G protein coupling in TP and IP. Significant challenges exist in expressing and purifying GPCR-CAMs in amounts required to pursue biophysical studies. Using tetracycline inducible HEK293S system, A160T was expressed at high-levels and CD spectropolarimetry studies were successfully pursued on the purified A160T. The CD spectra showed that the loss of thermal stability of the A160T mutant is due to the subtle changes in the secondary structure of the A160T protein. These studies involving molecular, biochemical and pharmacological approaches provide novel insights into the structure and function of prostanoid receptors TP and IP.

Microbiome-derived trimethylamine N-oxide (TMAO) has been associated with platelet hyperreactivity and subsequent atherogenesis. Whether physiological TMAO-levels influence plateletderived lipid mediators remains unknown. Little is known about pre-analytic factors potentially influencing TMAO concentrations. We aimed at developing a quantitative LC-MS/MS method to investigate in-vivo and in-vitro pre-analytical factors in TMAO analysis to properly assess the proposed activating effect of TMAO on platelets. TMAO, betaine, carnitine, and choline were analyzed by HILIC-ESI-MS/MS within 6 min total run time. Method validation included investigation of reproducibility, recovery, sensitivity, and in-vitro pre-analytical factors. A 24-h monitoring experiment was performed, evaluating in-vivo pre-analytical factors like daytime or diet. Finally, the effects of different TMAO concentrations on platelet activation and corresponding alterations of plateletderived eicosanoid release were analyzed. The method showed high reproducibility (CVs 5.3%), good recovery rates (96–98%), and negligible in-vitro pre-analytical effects. The influence of in-vivo pre-analytical factors on TMAO levels was not observable within the applied experimental conditions. We did not find any correlation between TMAO levels and platelet activation at physiological TMAO concentrations, whereas platelet-derived eicosanoids presented activation of the cyclooxygenase and lipoxygenase pathways. In contrast to previously published results, we did not find any indications regarding diet dependency or circadian rhythmicity of TMAO levels. Our results do not support the hypothesis that TMAO increases platelet responsiveness via the release of lipid-mediators.

La correction de l'anémie par l'érythropoïétine recombinante humaine (rhEPO) en insuffisance rénale chronique (IRC) s'accompagne d'une augmentation de la pression artérielle. Nos travaux antérieurs indiquent que l'rhEPO accentue la dysfonction endothéliale qui est présente en IRC en augmentant la production d'endothéline-1 (ET-1). Par contre, en condition rénale normale, la pression artérielle et la concentration vasculaire d'ET-1 demeurent inchangées suite au traitement à l'rhEPO. Ces résultats suggèrent que l'rhEPO exerce un effet différent sur la production des hormones vasoactives telles la thromboxane A2 (TXA2) et la prostacycline (PGL) et sur le système ET-1 en IRC et chez l'animal normal. Notre étude démontre que l'administration d'rhEPO en IRC s'accompagne d'une augmentation supplémentaire de TXA? alors que le niveau de PGL demeure inchangé. Le ratio TXA2/PGL est donc augmenté ce qui suggère que la production de PGL est insuffisante pour contrer les effets de la TXA2. En effet, le blocage de la synthèse de TXA2 avec le ridogrel prévient l'hypertension artérielle induite par l'rhEPO en urémie alors que le blocage simultané de la TXA2 et de la PGI2 avec l'acide acétylsalicylique est inefficace. Ces résultats soulignent l'importance de préserver la production de PGI2 lors de l'administration d'rhEPO en IRC. Chez l'animal normal, nous avons observé que l'administration d'rhEPO n'affecte pas la pression artérielle et la concentration vasculaire d'ET-1. Nous avons alors émis l'hypothèse que le récepteur (R) ETB (impliqué dans la clairance) pourrait jouer un rôle compensateur sur la pression artérielle et la clairance de l'ET-1 lors de l'administration d'rhEPO chez l'animal normal. En effet, nos résultats démontrent que l'expression de PARNm du récepteur ETH est augmentée ainsi que sa densité sur l'endothélium des vaisseaux d'animaux traités à l'rhEPO. Afin de démontrer l'effet compensateur du récepteur ETB, nous avons administré de l'rhEPO à des souris ETBR hétérozygotes <+) , ETA<+/_) et sauvages. Le traitement à l'rhEPO cause une augmentation significative de la pression artérielle et de la concentration vasculaire d'ET-1 seulement chez les souris ETBR(+/) . Ainsi, ces résultats démontrent que l'rhEPO peut moduler le système ET1/ETHR en condition normale. Cette augmentation de l'expression du récepteur ETB pourrait contribuer à maintenir la pression artérielle lors de l'administration d'rhEPO à des animaux normaux. À l'inverse, des conditions où le récepteur ETB est déficitaire, comme chez les souris ETBR ( + ) et lors de ITRC, pourraient entraîner une augmentation de la pression artérielle lors de l'administration d'rhEPO. Des analyses de l'expression de gènes par microarray ont aussi été menées sur les aortes de ces rats. Les résultats révèlent que l'effet de l'rhEPO sur l'expression des gènes est différent entre les rats normaux et urémiques. En effet, l'analyse de ces données suggère que des gènes comme la superoxyde dismutase et la glutathionc peroxydase pourraient être impliqués dans l'hypertension artérielle induite par l'rhEPO en IRC. Ces résultats permettent d'identifier de nouvelles avenues de recherche qui permettront d'élucider les mécanismes physiopathologiques de cette forme iatrogénique d'hypertension artérielle. En conclusion, mes travaux de recherche au doctorat révèlent que l'rhEPO exerce une action pléiotropique sur les vaisseaux sanguins et cette action est différente entre le rat normal et le rat urémique. Chez l'animal urémique, l'rhEPO accentue la dysfonction endothéliale déjà existante et l'hypertension artérielle alors que chez le rat normal, l'administration d'rhEPO est associée à des mécanismes compensatoires permettant de maintenir la pression artérielle à un niveau normal. Nos résultats fournissent des évidences d'un nouveau rôle modulateur de l'rhEPO sur les vaisseaux sanguins et permettront de développer des approches thérapeutiques plus spécifiques pour le traitement de l'hypertension artérielle induite par l'rhEPO en IRC. / The treatment of anemia with recombinant human erythropoietin (rhEPO) in chronic renal failure (CRF) induces hypertension. Our previous studies showed that rhEPO increases the existing endothelial dysfunction in rats with CRF by stimulating the production of endothelin-1 (ET-1). In contrast, in normal animals, blood pressure and vascular ET-1 concentrations remain unchanged following rhEPO therapy. These results prompted us to verify whether rhEPO exerts a different effect on the production of vasoactive hormones such as thromboxane A2 (TXA2) and prostacyclin (PGI2), and on the vascular ET-1 system in CRF and normal animals. Our study showed that rhEPO administration in CRF rats is associated with a further increase in vascular TXA2 concentrations, while prostacyclin (PGE) levels remained unchanged. The ratio of TXA2/PGI2 was increased, suggesting that PGI2 production is insufficient to oppose the effects of TXA2. Consistent with this finding, TXA2 synthesis blockade with ridogrel prevented rhEPO-induced hypertension in uremic rats while the inhibition of the synthesis of both TXA2 and PGI2 with acetylsalicylic acid was ineffective. These results stress the importance of preserving PGI2 production when treating rhEpoinduced hypertension in CRF. In normal animals, we observed that rhEPO administration does not affect blood pressure and vascular ET-1 concentrations. We hypothesised, that the ETB receptor (a clearance receptor) could play a compensatory role in maintaining ET-1 levels in normal animals under rhEPO treatment. In keeping with that, the vascular expression of the ETB receptor as well as its endothelial density were increased in normal rats receiving rhEPO. To further document the compensatory role of the ETB receptor (R), we administered rhEPO to ETBR heterozygous <+/) , ETAR <+/_) and wildtype mice. The administration of rhEPO caused an increase in blood pressure and vascular ET-1 concentrations in ETBR mice exclusively. These results indicate that rhEPO can modulate the endothelial ET-1/ETBR system in normal conditions. This increase in endothelial expression of the ETBR may contribute to maintaining normal blood pressure during rhEPO administration in normal animals. In contrast, conditions with deficient ETBR expression, such as in ETBR( + ' mice orCRF rats, may lead to hypertension while receiving rhEPO therapy. Microarray gene expression analyses were also performed on the aorta of these rats. We observed that the effect of rhEPO on gene expression is different between normal and uremic rats. Indeed, the results suggest that genes such as superoxide dismutase and glutathione peroxydase might be involved in hypertension induced by rhEPO in CRF. These results pave the way toward new research directions in elucidating the physiopathogenic mechanisms of this iatrogenic form of hypertension. In conclusion, my doctoral studies reveal that rhEPO exerts a pleiotropic action on blood vessels and this action is different between normal and CRF animals. In uremic conditions, the administration of rhEPO aggravates the existing endothelial dysfunction and hypertension whereas in normal animals there are compensatory mechanisms that contribute to maintaining blood pressure normal. Our results provide further insights into novel modulatory actions of rhEPO on blood vessels which are highly relevant to the development of more specific and rational approaches for the management of rhEPOinduced hypertension in CRF.

Le diabète de type-2 et le syndrome métabolique sont associés à une augmentation du stress oxydant et du risque cardiovasculaire. L'hyperactivation plaquettaire et les dyslipoprotéinémies sont deux causes majeures de l'athérothrombose. Nous avons montré que des lipoprotéines de basse densité (LDL) issues du plasma de diabétiques de type-2 activent les plaquettes sanguines. L'objectif principal de notre étude a été d'établir le profil en lipides et peroxydes lipidiques de LDL provenant de volontaires ayant un syndrome métabolique (SM), un diabète de type-1 (DT-1) ou de type-2 (DT-2), comparativement à celui de volontaires sains (V). Un autre objectif a été de déterminer leur impact sur l'activation plaquettaire. Seules les LDL des groupes SM et DT-2 ont des anomalies lipidiques telles que : augmentation des triacylglycérols, diminution des esters de cholestérol et de l'acide linoléique. Les LDL des groupes SM, DT-1 et DT-2 présentent un stress oxydant, démontré par l'augmentation des produits de peroxydation lipidique comme les acides gras hydroxylés et le dialdéhyde malonique, ainsi que par la diminution des plasmalogènes (sous-classe de phospholipides à éthanolamine). Comparativement aux plaquettes incubées avec les LDL de V, les plaquettes incubées avec les LDL des autres groupes sont activées comme le montre une exacerbation de la cascade de l'acide arachidonique (p38 MAPK, phospholipase A2 cytosolique, thromboxane A2). Ainsi, dans les états pré-diabétique et diabétique de type-2, les LDL subissent des modifications lipidiques et oxydatives, puis activent les plaquettes. Nos résultats suggèrent que les peroxydes lipidiques des LDL induisent l'hyperactivation plaquettaire.