Hemodynamics (the patterns of blood flow along blood vessels), such as laminar shear stress (LSS) and oscillatory shear stress (OSS), plays an important role in maintaining vascular homeostasis and also the pathogenesis of atherosclerosis. LSS, occurring mainly in the straight part of the vascular tree, is protective against the formation of atherosclerotic plagues, while OSS is the predominant hemodynamic pattern experienced by the branched regions and curvatures in the vascular system, in which most of atherosclerotic plaques are developed. Recent evidence indicates that the Hippo pathway is important in transducing mechanical stimulation. However, the exact role of Hippo signaling in hemodynamics and the development of atherosclerosis is unclear. Therefore, the present study was designed to investigate how the Hippo pathway responds to hemodynamic stimulation and the impact of this pathway in the development of atherosclerosis. / The central axis of the Hippo pathway consists of a chain of kinase cascade including serine/threonine-protein kinase 1/2 (LATS1/2) and downstream YAP/TAZ effectors. Activation of the Hippo pathway leads to phosphorylation, nuclear exportation and inhibition of YAP/TAZ transcription factors. The present study is probably for the first time to demonstrate that the Hippo pathway can be activated in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) subjected to LSS. Western blotting results showed that Hippo activation was most likely mediated by phosphorylation of Hippo kinase YAP. The results from the nuclear fractioning assay showed that the amount of the nuclear fraction of YAP/TAZ was reduced in HUVECs subjected to LSS. Repression of the established YAP/TAZ target genes CTGF and CYR61, and inhibition of Hippo reporter gene after LSS treatment, further supported the notion that activation of the Hippo pathway inhibits the YAP/TAZ downstream genes expression. In vivo experiments showed an increased YAP phosphorylation in the straight area of C57 mouse thoracic aortas as compared with aortic arch, which favors that LSS activates Hippo pathway under the physiological condition. To further understand whether different flow patterns have different impacts on the Hippo pathway, the effects of OSS were also studied. As expected, OSS inhibited the Hippo pathway through inducing the de-phosphorylation of YAP, and promoting the nuclear localization of YAP/TAZ. The induction of YAP/TAZ target genes (CTGF and CYR61) further confirms that OSS-induced inhibition of the Hippo pathway enhanced the YAP and TAZ transcription activity. / Since unfavorable hemodynamics is among the most important factors in the development of atherosclerotic plaques, I hypothesized that inhibition of the Hippo pathway is involved in the formation of atherosclerotic plaques. Adhesion molecules are a group of cell surface proteins that mediate cell-cell interaction. During the initiation phase of atherosclerosis, adhesion molecules are highly expressed on the surface of endothelial cells to enhance the attachment of monocytes to the vascular wall, leading to monocyte accumulation within the vascular wall. OSS up-regulates the expression of several adhesion molecules; however, the underlying mechanisms remain elusive. To identify the role of Hippo pathway in adhesion molecules expression, the gene expression of HUVECs with YAP/TAZ knockdown was investigated using real-time PCR. The expression of the four adhesion molecule, ICAM1, E-Selectin, MCP1 and CXCL1 were down-regulated by YAP and TAZ gene silencing. To further study the mechanism, the promoter region of the four target genes were amplified from HUVECs genomic DNA, and subcloned into PGL3 reporter plasmids. The promoter activity was tested by co-transfection of the reporter plasmids with different dosages of constructively active YAP and TAZ. The present results showed that activated YAP and TAZ dose-dependently increased the promoter activity of these four genes. CXCL1 was selected for further examination because it has been recently reported to play an important role in oxidized LDL-induced elevation of monocyte attachment to endothelial cells. Analysis of the CXCL1 promoter revealed that two TEAD1 binding sites located within the promoter region of CXCL1. The reporter gene assay results showed that TEAD1 induced the CXCL1 reporter activity, indicating that the CXCL1 promoter was regulated by YAP/TAZ/TEAD complex. The EMSA analysis further demonstrates that constitutively active YAP and TAZ induced the binding between TEAD1 and the CXCL1 promoter, suggesting a direct association between TEAD1 and CXCL1 promoter. / To further explore whether the Hippo pathway activation could regress atherosclerosis development, TAZ knockdown adenovirus was generated and administered to ApoE⁻/⁻ mice fed on Western diet. The formation and sizes of atherosclerotic plaques were visualized using oil red O staining. Monocyte infiltration was estimated using monocyte marker CD68. The expression of CXCL1 was detected using immunohistochemistry. The present results demonstrated that TAZ gene silencing significantly suppressed the expression of CXCL1 and reduced the sizes of atherosclerosis plagues as well as monocyte infiltration in the vascular wall of ApoE⁻/⁻ mice. / To further identify whether Hippo pathway could be a drug target for the treatment of atherosclerosis, several known atherosclerosis risk factors and beneficial factors were tested for their effects on Hippo pathway. Four out of nine atheroprotective factors were found to suppress the TEAD reporter activity. On the other hand, four out of six risk factors were identified to increase the TEAD reporter activity. The present results suggest that atheroprotective LSS activates while atherogenic OSS inhibits the Hippo pathway. / The Hippo pathway could be activated by atheroprotective LSS, while it was inhibited by atherogenic OSS. Activation of the Hippo pathway inhibits the development of atherosclerosis at least in part through reducing the expression of adhesion molecules and decreasing macrophage accumulation in the vascular wall. The present new findings suggest that targeting this Hippo pathway is potentially useful for therapeutic intervention of atherosclerosis and associated vascular diseases. / 血流動力學（即血液在心血管系統中流動的模式），包括層流剪切應力（LSS）和振盪剪切應力（OSS）。血流動力學在血管穩態和動脈粥樣硬化的發展中起重要作用。LSS主要發生在血管系統的直線部分，可防止動脈粥樣硬化斑塊形成，而OSS是血管系統中分支和彎曲部分的主要血流模式，大部份粥樣硬化斑塊發生在此。最近的研究表明，Hippo通路在機械刺激傳導中起重要作用。然而，Hippo信號通路在血流動力學和動脈粥樣硬化發展中的確切作用目前尚不清楚。因此，本論文的目的是研究Hippo通路如何響應血流動力學刺激以及Hippo通路對動脈粥樣硬化的發展的影響。 / Hippo通路主要由一系列激酶鏈和下游YAP/ TAZ效應器組成，其中的激酶鏈包括絲氨酸/蘇氨酸－蛋白激酶1/2（LATS1/ 2）。激活Hippo通路可導致YAP和TAZ磷酸化，促進其出核運輸並抑制其轉錄激活的功能。本研究中，我們首次證明了在LSS條件下，人臍靜脈內皮細胞（HUVEC）和人主動脈內皮細胞（HAECs）中的Hippo通路被激活。Western雜交結果表明，Hippo通路的激活很可能是受YAP激酶的磷酸化調節。細胞核質分離結果顯示在LSS條件下，人臍靜脈內皮細胞中YAP/ TAZ的細胞核部分的量顯著減少。LSS处理后，YAP/ TAZ靶基因CTGF和CYR61的表達降低，其TEAD告基因受到抑制，这進一步證明激活Hippo通路可抑制YAP/ TAZ下游基因的表達。動物實驗結果顯示YAP磷酸化和YAP/ TAZ出核在C57小鼠的胸主動脈直的區域顯著高於彎的區域，這也表明在生理條件下，LSS可激活Hippo通路。為了進一步了解不同的血流模式是否對Hippo通路產生不同的影響，我們研究了OSS條件下的Hippo通路。正如所期，OSS通過誘導YAP的去磷酸化，促進YAP/ TAZ的核內分佈，從而抑制Hippo通路的活性。OSS誘導YAP/ TAZ靶基因（CTGF和CYR61）產生轉錄活性，證明了OSS可抑制Hippo通路。 / 不利的血流模式是動脈粥樣硬化斑塊產生的最重要因素之一，因此我們推測抑制Hippo通路或許可導致動脈粥樣硬化斑塊的形成。粘附分子是一類調節細胞間相互作用的細胞表面蛋白。在動脈粥樣硬化形成的初始階段，粘附分子會在內皮細胞的表面高表達，從而提高單核細胞附著和聚集於血管壁上。OSS上調粘附分子的表達，但機制仍不清楚。為了識別Hippo通路在粘附分子表達中的作用，本文用實時定量PCR檢測了YAP/ TAZ敲低了的人臍靜脈內皮細胞的基因表達水平。實驗結果顯示四種粘附基因，包括ICAM1，E-Selectin，MCP1和CXCL1通過YAP和TAZ的基因沉默而被下調。為了進一步研究此中機制，本文擴增了臍靜脈內皮細胞的基因組DNA中的四個靶基因啟動子區，並將其克隆到PGL3報告質粒。通過共轉染不同濃度的組成型激活的YAP和TAZ，本文測定了這四個啟動子的活性。結果顯示激活的YAP和TAZ能夠劑量依賴性上調這四個基因的啟動子活性。由於最近有報導說氧化低密度脂蛋白可誘導單核細胞附著於內皮細胞，而CXCL1在此誘導過程中具有重要作用，因此本文選擇CXCL1作進一步研究。通過分析CXCL1的啟動子，本文發現CXCL1的啟動子區域內有兩個TEAD1結合位點。報告基因檢測結果顯示TEAD1可誘導CXCL1報告活性，這表明CXCL1啟動子是通過YAP/ TAZ/ TEAD複合體來調節。EMSA分析進一步揭示了組成型激活的YAP和TAZ可誘導TEAD1和CXCL1啟動子之間的結合，表明TEAD1直接結合到CXCL1啟動子。 / 為進一步開拓Hippo通路是否是動脈粥樣硬化消退潛在的治療靶點，本文構建了TAZ敲低的腺病毒並將其轉入餵以西方飲食的ApoE⁻/⁻小鼠。通過油紅O染色來觀察動脈粥樣硬化斑塊的形態和大小。用單核細胞表面標記物CD68測定單核細胞對血管的浸潤。用免疫組化鑑定CXCL1的表達。結果表明TAZ基因沉默顯著抑制ApoE⁻/⁻小鼠血管壁CXCL1的表達，降低動脈粥樣硬化斑塊的大小及單核細胞浸潤。 / 為了進一步確定Hippo途徑是動脈粥樣硬化的藥物治療靶點，本文對幾個已知的動脈粥樣硬化的危險因子和有利因子對Hippo通路活性的影響進行了研究。出人意料的是，九個動脈粥樣硬化的保護因子中，有四個被確定為抑制TEAD報告基因活性。另一方面，六個危險因子中，有四個被鑑定為可提高TEAD報告活性。這些結果與上面的研究發現共同證明了LSS降低動脈粥樣硬化，激活Hippo通路，而OSS促進動脈粥樣硬化，抑制Hippo通路。 / 本研究確定了層流剪應力（LSS）激活Hippo通路，抑制動脈粥樣硬化；振盪剪切應力（OSS）抑制Hippo通路，導致動脈粥樣硬化。Hippo通路被激活能夠抑制動脈粥樣硬化的機制是通過降低粘附分子的表達，減少巨噬細胞在血管壁上的附著。這一新發現表明，靶向Hippo通路對介入治療動脈粥樣硬化以及相關的血管疾病具有潛在的應用意義。 / Wang, Li . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 129-138). / Abstracts also in Chinese. / Title from PDF title page (viewed on 08, November, 2016). / Wang, Li. / 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. / Detailed summary in vernacular field only.

[Truncated abstract. Please see the pdf format for the complete text.] Interest in the role of peroxynitrite in the pathogenesis of atherosclerosis has increased due to many in vitro studies which have demonstrated its potent oxidising and nitrating capability and immunohistochemical staining studies which demonstrate nitration of tyrosine in vivo. It is frequently suggested that the production of nitric oxide and superoxide at sites of inflammation implicates peroxynitrite as the major damaging reactive nitrogen species in vivo. Evidence for a role for peroxynitrite is often demonstrated by measurement of 3-nitrotyrosine yet even this cannot distinguish peroxynitrite from other nitrating species. Clearly, however, if peroxynitrite is important in atherogenesis, then identification of mechanisms for its detoxification could provide a means of preventing such effects. Therefore, this Thesis has sought to determine whether phenolic compounds of dietary origin can be preferentially nitrated by reactive nitrogen species thereby protecting endogenous structures, such as low density lipoproteins, from atherogenic modifications. This Thesis focuses upon phenolic acids as they have received relatively less attention than other classes of phenolic compounds, such as flavonoids, yet they are quite abundant in socially important beverages such as red wine. In order to complete the required analyses, the development of methods to detect phenolic acids and their nitration products together with 3-nitrotyrosine, dityrosine and 5-nitro-γ-tocopherol was necessary. The initial in vitro experiments described herein sought to determine the products of reaction of peroxynitrite with phenolic acids of the 4-hydroxy and 3,4-dihydroxy type and then to examine whether these products could account for a protective effect upon tyrosine, lipids and endogenous anti-oxidants, if any was observed, when isolated LDL was treated with SIN-1, which releases peroxynitrite through the simultaneous generation of nitric oxide and superoxide. A concurrent minor focus was to examine the relationship between structure and activity of these phenolic acids under various regimes of oxidative insult. These experiments indicate that, at least in this in vitro model, oxidation is a dominant mechanism over nitration. Peroxynitrite was shown to nitrate coumaric acid in moderate yields but exclusive oxidation of caffeic acid appeared to occur. Although a potential role for γ-tocopherol as an anti-nitration agent was inferred, all types of chemical treatment of LDL in the presence of phenolic acids yielded oxidation as the primary end point. In fact, nitration of tyrosine was not detected and nitration of coumaric acid was at the limit of detection. Since nitration of tyrosine is generally regarded as important in many disease states, a more physiological nitrating mechanism involving artificially stimulated neutrophils was used. This system demonstrated that although physiologically relevant reactive nitrogen species can result in nitration of phenolic compounds, in a complex system including biological structures (LDL) and phenolic compounds, oxidation but not nitration of all species appears to occur. As a consequence of the results above, an examination of carotid plaque was undertaken to determine to what extent nitration occurred relative to oxidation in atherosclerotic tissue. These studies applied methods developed herein to detect 3-nitrotyrosine and dityrosine in complex biological matrices as markers of nitration and oxidation respectively. The data obtained demonstrated that nitration was a minor modification of protein (0.01%) compared to oxidation (0.3%) even in a highly diseased tissue such as carotid artery plaque. A secondary study examining plasma revealed that dityrosine, which has been implicated in irreversible albumin aggregation in chronic renal failure and more recently in heart disease, is elevated in chronic renal failure subjects compared to well matched controls. A separate examination of plasma from healthy subjects revealed that in both the fasting and post prandial state 3-nitrotyrosine could not be detected and, in fact, interfering species could be problematic in the GC-MS analysis of 3-nitrotyrosine. The lack of nitration of any substrate observed in vitro using reactive nitrogen species generated in the aqueous phase, the relative lack of nitration of tyrosine in plaque proteins and the lipophilicity of nitric oxide, the precursor of all reactive nitrogen species, suggested that nitration could be more closely associated with lipid structures. The known ability of γ-tocopherol to form 5-nitro-γ-tocopherol was used to probe this concept. The 5-nitro-γ-tocopherol content of lipid extracts obtained from carotid artery plaques was very high (30%). This indicated that nitration is predominantly a lipid phase phenomenon and that nitrating species are present in much greater abundance than oxidising species in vivo.

Abstract not available

[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Atherosclerosis is a common degenerative disease in which the clinical manifestations are often through stroke or myocardial infarction. Some of the established risk factors for atherosclerosis include elevated plasma low-density lipoprotein (LDL)-cholesterol levels, obesity, diabetes mellitus (DM) and cigarette smoking. Of the risk factors, an elevation in plasma LDL is one of the most established and the most researched. This is partly a consequence of the deposition of cholesterol within arterial intima being a crucial step in the progression of atherosclerosis, combined with the finding that LDL particles are a major transporter of cholesterol in circulation. Recently there is increasing evidence showing a role of the other major transporter of cholesterol in circulation, chylomicron remnants, in the progression of atherosclerosis. The notion of atherosclerosis as a postprandial phenomenon has been further substantiated by the emergence of evidence showing a direct role of chylomicron remnants in arterial cholesterol deposition. Based on evidence that chylomicron remnants are proatherogenic, the suggestion arises that accumulation of postprandial lipoproteins in plasma may add another dimension of risk to the development of coronary artery disease (CAD). This thesis tests the general hypothesis that individuals with or at high risk of CAD have postprandial dyslipidaemia and that this metabolic abnormality is correctable with a class of lipid-lowering drugs called statins. To test the hypothesis, clinical studies were conducted in normolipidaemic CAD patients, heterozygous familial hypercholesterolaemia (FH) and postmenopausal women with type 2 DM. Determination of postprandial dyslipidaemia by comparison with control populations were conducted initially in each patient group (Studies 1, 3 and 5), followed by intervention studies investigating possible modulation of the dyslipidaemia with a statin (Studies 2, 4 and 6). Six observation statements based on case-control comparisons of postprandial lipaemia in patients with or at risk of CAD and the effects of statins on postprandial dyslipidaemia in the patient groups were derived from the general hypothesis. The observation statements were examined in the individual studies described below. Postprandial lipoprotein metabolism was assessed using a number of methods. For comparison of postprandial lipaemia in Studies 1 and 2, a classic oral fat challenge was utilised. As markers of chylomicrons and chylomicron remnants, retinyl palmitate and triglyceride were measured postprandially as well as apolipoprotein (apo) B48 concentrations, a specific marker of intestinal lipoproteins. ApoB48 was also measured in the fasting state and found to predict the postprandial responses of retinyl palmitate, triglyceride and apoB48. This suggested that fasting measurement of apoB48 could be used as a simple indicator of postprandial dyslipidaemia. Consequently for Studies 3 - 6, fasting apoB48 measurements were used as primary markers of postprandial dyslipidaemia. Other markers for chylomicrons and their remnants utilised were fasting plasma concentrations of remnant-like particle-cholesterol (RLP-C) and apoC-III. As well as these static markers, chylomicron remnant catabolism was measured using a stable isotope breath test. The breath test involves the intravenous injection of a chylomicron remnant-like emulsion labelled with ¹³C-oleate and measurement of enriched ¹³CO2 in expired breath by isotope ratio mass spectrometry. The fractional catabolic rate (FCR) of the injected emulsion was subsequently calculated using multi-compartmental modeling (SAAM II). The studies are presented in this thesis as published and unpublished works. In Study 1, postprandial lipoprotein metabolism was compared between 18 normolipidaemic CAD patients (cholesterol 4.54 ± 0.12 mmol/L, triglyceride 1.09 ± 0.16) with 13 asymptomatic healthy controls using an oral fat challenge. Normolipidaemic CAD patients had higher postprandial area-under-curve (AUC) for triglyceride (+34%, p=0.019), retinyl palmitate (+74%, p=0.032) and apoB48 (+36%, p<0.001). Fasting apoB48 was also higher (+41%, p=0.001) and found to correlate significantly with AUC of triglyceride (p=0.017), retinyl palmitate (p=0.001) and apoB48 (p<0.001). The data suggest that normolipidaemic CAD patients have increased concentrations of intestinal lipoproteins in the fasting and postprandial state. In addition to these findings, significant correlations of fasting apoB48 with postprandial markers (p<0.02) suggests the fasting marker to be a simpler surrogate marker for the degree of total postprandial lipaemia. Study 2 investigated the effect of atorvastatin treatment on postprandial dyslipidaemia found in the 18 near-normolipidaemic CAD patients from Study 1. The trial was conducted in a randomised, placebo-controlled design, using oral fat challenges before and after 12-weeks atorvastatin/placebo treatment. Compared with the placebo group, atorvastatin decreased the total postprandial AUC for iii triglyceride (-22%, p=0.05) and apoB48 (-34%, p=0.013). Fasting markers of apoB48 (-35%, p=0.019) and RLP-C (-36%, p=0.032) also decreased significantly. Atorvastatin was also found to increase LDL-receptor activity by +218% (p<0.001) as reflected in binding studies. The data suggest atorvastatin reduces the fasting levels of intestinal lipoproteins as well as total postprandial lipaemia, but without acute dynamic changes in postprandial lipaemia. The reduction in fasting and total postprandial lipoprotein levels could be partly attributed to an increase in LDL-receptor mediated removal from circulation. In Study 3, postprandial lipaemia was compared in 15 heterozygous FH patients with 15 healthy controls. FH patients had higher fasting concentrations of apoB48 (+56%, p<0.001) and RLP-C (+48%, p=0.003). The elevation in these fasting markers of chylomicrons and their remnants suggests FH patients have postprandial dyslipidaemia due to an accumulation of these particles in plasma. Study 4 examined the effects of long- (> 6 months) and short-term (4 weeks) simvastatin treatment on modulating postprandial dyslipidaemia found in the 15 FH patients from Study 3. Short- and long-term simvastatin treatment decreased the fasting concentrations of apoB48 (-29% and 15% respectively, p<0.05) and RLP-C (both -38%, p<0.001), but did not significantly alter the FCR of the injected chylomicron remnant-like emulsion. The data suggest that in heterozygous FH both long- and short-term simvastatin treatments decrease the fasting markers of postprandial lipoproteins by mechanisms that may not be mediated via processes differentiated by the 13CO2 breath test. This implies that the effect on postprandial lipaemia may be from a decrease in production and/or a possible increase in catabolism of triglyceride-rich lipoproteins (TRLs). In Study 5, postprandial lipaemia was compared in 24 postmenopausal women age and body mass index matched with 14 postmenopausal women with type 2 DM. Postmenopausal diabetic women were found to have higher fasting concentrations of apoB48 (+21%, p=0.021) and apoC-III (+16%, p=0.042) as well as lower FCR of the chylomicron remnant-like emulsion (-50%, p<0.001). The data suggest that postmenopausal diabetic women have postprandial dyslipidaemia, and that this is due to delayed catabolism of chylomicron remnants. Study 6 was an hypothesis-generating exercise examining the effects of 4-weeks pravastatin treatment on postprandial dyslipidaemia found in 7 postmenopausal women with type 2 DM from Study 5. Although plasma LDL-cholesterol was reduced (-19%, p=0.028), there were no significant effects found on fasting apoB48 concentrations (-12%, p=0.116) or the FCR of the chylomicron remnant-like emulsion (+38%, p=0.345). A larger sample size of patients and/or treatment with a more potent statin at a dosage known to affect chylomicron remnant metabolism would be required to demonstrate a significant reduction in postprandial dyslipidaemia in postmenopausal women with type 2 DM. The results of the above mentioned studies combined support the general hypothesis that postprandial dyslipidaemia is a feature of patients with or at risk of CAD. This defect may be demonstrated using fasting apoB48 as an indicator of the degree of postprandial lipaemia. Postprandial dyslipidaemia may reflect a reduction in catabolism, as suggested with the breath test in type 2 DM, and/or an over overproduction of chylomicrons. Both these mechanisms would also increase competition for lipolysis and clearance pathways between hepatically and intestinally-derived lipoproteins. The exact mechanisms by which postprandial dyslipidaemia occurs are yet to be determined. Statins appear to improve defective postprandial lipaemia in patients with or at risk of CAD, which is in agreement with the general hypothesis. The effectiveness of a statin is dependant on their potency in inhibiting cholesterol biosynthesis and increasing receptor mediated clearance of LDL and chylomicron remnants. The studies conducted in this thesis show that postprandial dyslipidaemia can be reduced by statins but not to the extent demonstrated in controls. However, the demonstrated reduction in fasting and total postprandial lipaemia translates to a lowering in overall arterial exposure to circulating proatherogenic particles. The elevation in fasting and postprandial levels of proatherogenic chylomicron remnants found in the patient groups described in this thesis indicates another dimension to their risk of coronary disease. The reductions in the overall levels of proatherogenic particles in patients with or at high CAD risk, infers a possible reduction in the risk of coronary disease in these patients.