published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Master of Medical Sciences

Cigarette smoke (CS) is a well-established risk factor to cardiovascular health and the most preventable cause of death. Countless studies have demonstrated its harm to health and many more studies investigating its pathogenic mechanisms. While the CS-induced pathogenic mechanism of cardiovascular dysfunction has been mainly attributed to a combination of oxidative imbalance, vascular endothelial dysfunction, inflammation and modification of lipid profile, the focus of the current study was on the mediators of inflammation and the activation of signal pathways. In this study, we investigated the effects of CS on the pro-inflammatory/anti-inflammatory status in the heart and to elucidate the activation of specific signaling pathways in an in vivo rat model. Male Sprague-Dawley rats were divided into groups of CS exposure and sham air (SA) and exposed to 1 hour of respective CS and SA exposure daily for 56 days. The rats were then sacrificed and the ventricular homogenates were examined. Cardiac pro- inflammatory and anti-inflammatory mediators such as C-reactive protein (CRP), interleukin (IL)-6, cytokine-induced neutrophil chemoattractant (CINC-1), transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF) and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA) and the activation of specific signaling pathways was determined by Western blot analysis. CS caused suppression of cardiac CRP, IL-6, TGF-β1, and IL-10 and elevation of VEGF, revealing the imbalance of pro-inflammatory/anti-inflammatory status. Nuclear factor-κB (NF-κB) was also activated along with the activation of extracellular-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) but not p38 mitogen-activated protein kinase (MAPK) after 56 days of CS exposure. These data suggests the presence of a local adaptive mechanistic response to modulate cardiac pro-inflammatory/anti-inflammatory status via NF-κB/MAPK pathways after exposure to CS. These findings shed insight into the mechanistic pathways of CVD progression, allowing possible identification of selected mediators as biomarkers that could benefit early detection of CVD arisen from cigarette smoking. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

The endothelium is important for the regulation of vascular tone. In diseases like hypertension, the endothelial cells become dysfunctional. This dysfunction is characterized by nitric oxide (NO) deficiency, impairment of endothelium-dependent hyperpolarization (EDH) and the overwhelming production of endothelium-derived contracting factor (EDCF). Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme, producing carbon monoxide(CO), bilirubin and free iron. Up-regulation of the inducible isoform (HO-1) of the enzyme lowers blood pressure in animals. The purpose of the present study was to investigate whether or not up-regulation of HO-1by the pharmacological agent hemin improves endothelial function in arteries of spontaneously hypertensive rats(SHR). Twenty four hours after intraperitoneal injection of hemin (50mg/kg) in 36 weeks old SHR, the expression and activity of HO-1 were augmented, in both the endothelium and vascular smooth muscle. Hemin-treatment potentiated endothelium-dependent relaxations to the muscarinic agonist acetylcholine in both the aorta and the mesenteric artery, whereas the HO inhibitor protoporphyrin IX zinc (II) (ZnPP; 30 mg/kg) prevented the beneficial effect of hemin, suggesting that HO-1 induction improves endothelial function. Hemin-treatment did not augment acetylcholine-induced NO-mediated relaxations, and did not alter the expression level of either phosphorylated eNOS (Ser1177) or total eNOS, suggesting that the improvement of endothelial function by HO-1 induction cannot be attributed to an increased bioavailability of NO. In the mesenteric arteries, hemin treatment potentiated acetylcholine-evoked EDH-mediated relaxations in the presence of L-NAME and indomethacin. The IKCa channel blocker TRAM-34andthe Na+-K+-ATPase blocker ouabain significantly impaired these hemin-potentiated relaxations. NS309-induced TRAM-34-and ouabain-sensitive relaxations were enhanced by hemin-treatment. K+-induced ouabain-sensitive relaxations and the expression of Na+-K+-ATPase were increased by hemin-treatment. Taken in conjunction, these observations imply that the improved EDH-mediated relaxations by HO-1 induction is due to an improvement of IKCa-Na+-K+-ATPase pathway. Treatment with an antioxidant apocynin (50mg/kg) showed a similar effect as hemin, and the combined treatment with hemin and apocynin did not cause a greater improvement. In vitro treatment with bilirubin, enhanced EDH responses and K+-induced ouabain-sensitive relaxations. These observations suggest that the effect of HO-1 induction on EDH-mediated relaxations is possibly due to its antioxidant properties and the production of bilirubin. In the aortae, hemin-treatment reduced endothelium-dependent contractions in response to acetylcholineor to a calcium ionophoreA23187. Production of reactive oxygen species (ROS) was suppressed by hemin-treatment, judging from the results of 2’,7’-dichlorodihydrofluoresein diacetate staining, dihydroethidium staining and lucigenin chemiluminescence, which was attributed to the decreased expressions of NADPH oxidase-2 (Nox2) and cyclooxygenase-1(COX-1). The production of prostacyclin was decreased, which was explained by a lower expression of COX-1. Contractions to vasoconstrictor concentrations of prostacyclin and its mimetic iloprost were attenuated, suggesting that the responsiveness of thromboxane-prostanoid receptors (TP receptors) to prostacyclin was decreased by hemin-treatment. The effects of HO-1 on the suppressed production of ROS and prostacyclin, and the decreased responsiveness of TP receptors, contribute to its inhibitory role on EDCF-mediated response. Thus, up-regulation of HO-1 improves endothelial function in the SHR by potentiating EDH response and impairing EDCF. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Alzheimer’s disease (AD) is the most frequent neurodegenerative disorder which is characterized by impaired mental functions such as memory, language, perception, behavior and personality, as well as cognitive skills. The molecular mechanisms underlying this disease is still largely unknown, but numerous evidence emerge to support a cell cycle hypothesis which implicates the deregulation of cell cycle proteins as key mediators of neuronal dysfunction and loss in AD brains. One of these signals in Aβ-induced neuronal death model is Cdk/Rb/E2F pathway, where Aβ insult evokes activation of Cdk4/6, which subsequently phosphorylates pRb protein, resulting in activation of E2F transcription factors. However, the mechanism(s) by which Cdk/Rb/E2F mediates neuronal death remains elusive. Therefore, the goal of this project is to characterize the downstream events of cell cycle pathway, which include the involvement of transcription factors c-Myb, Pax6 and Patz1 in Aβ-induced neuronal death signaling. In this study, we showed that Pax6 is a direct target gene for Both E2F1 and c-Myb. Both Pax6 and c-Myb are up-regulated by Aβ insults in cultured cortical neurons. And with E2F1 silencing by siRNA, Aβ-induced Pax6 and c-Myb expression is blocked, suggesting E2F1 is responsible for their elevation. Importantly, siRNA-mediated downregulation of either c-Myb or Pax6 protects neurons from death evoked by Aβ peptide, suggesting they are proapoptotic proteins, delivering death signals sent from upstream E2F1. Next, though ChIP assay, we identified two target genes for Pax6. One is Patz1, another transcription factor that is Aβ-induced pro-apoptotic protein. The other one is GSK3β, which is a pathogenic kinase involved in Tau protein hyperphosphorylation and NFT formation. In conclusion, this dissertation shows that cell cycle regulators Cdk/Rb/E2F modulate neuronal death signals by activating downstream transcription factors c-Myb and Pax6, further upregulating GSK3β. We provided evidence suggesting that Aβ induced neurotoxicity leads to Tau hyperphosphorylation through a mechanism involving cell cycle activation and subsequent activation of c-Myb/Pax6/GSK3β. In brief, in the present study, we delineate a transcriptional cascade downstream of cell cycle pathway leads to neuronal apoptosis as well as Tau/NFT pathology. The characterization of this novel pathway lends support for development of new therapeutic agents and for better experimental models for AD. Lastly, the cascade between cell cycle activation and tauopathy in Aβ-induced neuronal death needs to be further researched in the future. / HKU 3 Minute Thesis Award, Champion (2011) / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

 Retinopathy of prematurity (ROP) commonly occurs in premature babies, with the first phase of vessel cessation followed by a second phase of vessel proliferation. In addition to vascular changes, neuronal abnormalities have also been observed. However, evidence for morphological changes of retinal neurons at the cellular level is lacking. Oxidative stress has been highly indicated in the pathogenesis of ROP. Increased oxidative stress level was demonstrated in preterm babies expecially in those with ROP. The activity of aldose reductase (AR), the first enzyme in the polyol pathway, has been found to contribute to oxidative stress. Therefore, the role of AR in ROP was examined using a mouse model of oxygen-induced retinopathy (OIR), which was a well-established model to mimic human ROP. Studies in examining the effects of AR on retinal vasculature showed that genetic deletion or pharmacological inhibition of AR reduced vaso-obliteration and neovascularization, possibly through regulating VEGF-induced pathway. In addition, morphological changes of various retinal neurons at different time points in the mouse model of OIR were also demonstrated. The degree of effects from hyperoxic and hypoxic exposure appeared to depend on the different stages of maturation of various retinal neurons. AR deficiency showed protective effects on retinal neurons including horizontal cells, rod bipolar cells and amacrine cells, possibly through attenuating the damage on blood vessels as well as facilitating blood vessel re-growth in the avascular area which provide more nutrients and supply to the retinal neurons. To elucidate the protective role of AR deficiency in ROP, the changes in oxidative stress and oxygen-dependent gene expression including HIF-1α and iNOS were investigated. AR deficiency attenuated oxidative stress induction to protect the neonatal retina. In addition, AR deficiency also showed attenuated HIF-1α expression and enhanced iNOS expression. This served to strictly control the HIF-1α level which in turn can tightly regulate VEGF induction in the mouse retinae after OIR. In order to further elucidate the role of AR in the pathogenesis of ROP, effects of AR deficiency on glial cells and microglia were investigated. AR deficiency reduced retinal astrocytic activation in hyperoxia and induced early M?ller cell gliosis in hypoxia. In addition, AR deficiency enhanced the specific function of microglia in different areas with facilitation of revascularization in avascular area and promotion of tufts regression in neovascular area. Moreover, AR deficiency also reduced the activation of a key inflammatory mediator NF-κB, which was considered to contribute to neovascularization. Therefore, AR deficiency demonstrated regulatory roles in reponses of glial cells, microglia and inflammation, contributing to the protective effects on neonatal retina in the mouse model of OIR. Taken together, AR deficiency reduced the vascular and neuronal changes possibly through attenuating oxidative stress and glial responses as well as modulating inflammatory responses, indicating a beneficial role of AR inhibition in OIR. These findings highly suggest the therapeutic potential of AR inhibition in the treatment of ROP. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Breast cancer is the most common malignant cancer in western countries. It can be classified into various types/stages according to patient age, tumor size, histological grade or hormone receptor status. Obesity is a well-known risk factor of breast tumor. Studies have shown that overweight or obese postmenopausal women have a threefold higher risk to develop breast cancer in comparison to their lean or normal counterparts. There are many mechanisms that can link obesity with breast cancer and one of the major contributors is adipokines. The main focus of this study is adiponectin. Many cellular and animal studies have illustrated the inhibitory action of adiponectin on breast cancer cell proliferation. In this study, the effect of complete loss of adiponectin expression on breast cancer development in Mouse Mammary Tumor Virus-polyomavirus middle T antigen(MMTV-PyVT)mice [PyVT(+/-)]will be investigated. Mice with [ADN(+/+)]or without [ADN(-/-)] adiponectin gene were used for comparison. It was found that PyVT(+/-)ADN(-/-)mice had earlier tumor onset time and larger tumor volume than PyVT(+/-)ADN(+/+) mice. Histological analysis has demonstrated that increased and more dispersed metastasis existed in lung tissue of PyVT(+/-)ADN(-/-)mice in comparing with PyVT(+/-)ADN(+/+)mice. The aggressiveness of adiponectin deficient tumor was preserved after implantation into immune-deficient mice. Gene expression and protein expression studies of PyVT tumor have indicated a different expression level and pattern of two important molecules: P63 and YY1. In conclusion, tumor developed under microenvironment of adiponectin deficient will give rise to a more aggressive tumor. This tumor consistsof modified genotypes and phenotypes that are permanent and can be preserved after re-implantation into immuno-compromised mice. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

Background and objectives: A lot of studies proved that adipocyte fatty acid binding protein (A-FABP), an adipokine mainly expressed in adipocytes and macrophages, is the key link between obesity and inflammation which is suggested to be a therapeutic target for obesity-related diseases. Loss-of-function study was employed by using A-FABP knockout (KO) mice generated by our group to investigate role of A-FABP in high fat high cholesterol (HFHC) diet-induced obesity. Key findings: 1. Our study confirmed that HFHC diet-induced A-FABP KO mice have a significantly increased body weight when compared to the wild-type (WT) control mice. 2. Higher adiposity was the major reason for the A-FABP KO mice to be heavier than the WT controls under HFHC diet induction. 3. The marked increase of the weight of subcutaneous fat and peri-renal fat contributed to the higher adiposity of the HFHC-diet induced A-FABP KO mice when compared to the WT controls. 4. The HFHC-diet induced A-FABP KO mice significantly consumed less oxygen and produced less carbon dioxide suggesting the reduced energy expenditure but had higher weekly energy intake when compared with the WT controls, leading to higher adiposity. 5. The A-FABP KO mice were protected against HFHC diet induced glucose intolerance, insulin resistance, hyperglycemia and hyperinsulinemia when compared with the WT controls. There was also a better insulin secretion in response to glucose stimulation in A-FABP KO mice under prolonged HFHC diet induction when compared with the WT controls. 6. The A-FABP KO mice were protected against the development of hypercholesterolemia and hypertriglycemia when compared the WT controls under HFHC diet induction. However, there was no significant difference in the fasting serum free fatty acids (FFA) level among A-FABP WT and KO mice fed with standard chow (STC) or HFHC diet. 7. A-FABP KO mice were protected against isolated systolic hypertension (ISH) under HFHC diet induction. 8. The A-FABP KO mice were protected against HFHC diet-induced liver injury as indicated by a lower serum ALT level suggesting a better liver function when compared with the WT controls. 9. Under HFHC diet induction, M1 macrophage polarization was dominant in fat tissues of A-FABP WT mice but M2 macrophage polarization was dominant in fat tissues of A-FABP KO mice, suggesting an improved inflammatory status in the adipose tissue of the A-FABP KO mice when compared with the WT controls. This may also be the reason for why HFHC diet-induced A-FABP KO mice have an increased body weight but are metabolically healthier compared to their WT controls. Conclusions: A-FABP KO mice had a significant higher body weight and higher adiposity due to the reduced energy expenditure and increased weekly food intake as indicated in the metabolic cage study and the reason for metabolic healthier is due to the alleviated HFHC diet induced M1 macrophage polarization in various adipose tissues suggesting an improved inflammatory status in A-FABP KO mice comparing to the WT controls. / published_or_final_version / Medicine / Master / Master of Philosophy

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The basis for the species difference between B6C3F1 mice (susceptible) and Fischer 344 rats (resistant) to 4- vinylcyclohexene (VcH)-induced ovarian tumorigenicity was investigated. Greater than 95% of a single oral 400 mg/kg dose of [¹⁴C]VCH was eliminated in 48 hr by mice and rats. Approximately 50-60% of the administered dose was excreted in the urine, while the remaining 30-40% of the dose was expired as organically soluble radioactivity. VCH-treated mice had dramatically higher blood concentrations of the VCH metabolite VCH-1,2-epoxide compared to VCH-treated rats. Furthermore, mouse hepatic microsomes catalyzed the conversion of VCH to VCH-1,2-epoxide at greater rates than rat hepatic microsomes. The destruction of oocytes was used as an index of ovarian toxicity to compare the potency of VCH and VCH epoxides in the mouse and rat. VCH markedly reduced the number of small oocytes in mice while no detectable change in oocyte number occurred in rats. Epoxide metabolites of VCH destroyed oocytes in both species at lower doses than VCH. Inhibition of VCH epoxidation reduced VCH-1,2-epoxide blood levels and partially protected mice from VCH-induced ovarian toxicity. Thus, the conversion of VCH to epoxides and the subsequent destruction of oocytes are critical steps in the induction of ovarian tumors by VCH. Rats may be resistant because the amount of VCH converted to epoxides is insufficient to destroy oocytes. The biochemical basis for the species difference in the rate of VCH epoxidation by hepatic microsomes from mice and rats was investigated. studies using inducers and inhibitors of certain cytochrome(s) P450 showed that hepatic microsomes of female mice perform VCH epoxidation at greater rates than rats because of the constitutive expression of P450 IIA and lIB forms. Hepatic microsomes of human females perform VCH epoxidation at lower rates than rats. This suggests that if the rate of epoxidation of VCH by the liver is the most important factor determining susceptibility to VCH toxicity then the rat may better model the response of humans exposed to VCH than mice.