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Tetracycline molecules including doxycycline (DOX), consist of a group of broad-spectrum antibiotics. In addition, tetracyclines inhibit matrix metalloproteinase (MMPs) that contribute to tissue remodeling, inflammation, angiogenesis and are over-expressed in certain pathologies - such as Alzheimer’s disease, metastasis and diabetic foot ulcers (DFUs). Tetracyclines are hypothesized to inhibit MMPs through the chelation and sequestration of catalytic divalent ions such zinc and calcium. This inhibitory duality may be beneficial in pathologies that are characterized by MMP over-expression and prone to infection, such as DFUs. Compared to oral administration, topical DOX is an attractive route of administration for chronic wound healing as it may minimize the risks: associated antibiotic resistance; is being targeted directly to the wound bed. However, DOX is notoriously unstable in aqueous solution and common topical formulations. Liquid chromatography and mass spectrometry (LCMS) were employed to monitor stability using an in vitro MMP assay and an applicable E. coli anti-bacterial assay was assessed to quantify drug activity. 2 % (w/w) topical DOX demonstrated an acceptable stability 30 day when stored at 4 ºC. DOX inhibited MMP9 activity with an IC50 value of 48.27 μM. With respect to anti-bacterial activity, using cultured BL21 E.Coli and quantification of drug activity as an expression of colony forming units (CFUs) successfully reproduced the antimicrobial IC50 of doxycycline as 4.3 µM. Transdermal DOX has the potential to improve standard of care for DFUs, quality of life for the patient and reduce costs to the healthcare system. / Thesis / Master of Science (MSc) / Tetracyclines comprise of a group of broad-spectrum antibiotics; whose primary mechanism of action is inhibition of protein synthesis through binding of the bacterial ribosome. In addition, tetracyclines inhibit matrix metalloprotease (MMPs), zinc-dependent proteases that contribute to tissue remodeling, angiogenesis and are over-expressed in certain pathophysiologies such as diabetic foot ulcers (DFUs). The antibacterial mechanism of DOX on MMPs is reported and understood, however the inhibition is hypothesized to involve cation chelation. Thus, investigating this interaction is warranted to assist in developing a therapeutic for DFUs. A more logical product would involve direct topical application, such as a stable transdermal formulation of DOX.

Diabetic nephropathy (DN) affects around 40% of diabetic patients worldwide and has become a major health concern due to its high morbidity and mortality. The progression of DN is characterized by the thickening of glomerular basement membrane, albuminuria and the development of glomerulosclerosis. Renal function is eventually compromised. Due to various hemodynamic and metabolic changes, especially the elevated blood glucose level in diabetic patients, glomerular mesangial cells have been shown to upregulate transforming growth factor-β1 (TGF-β1) level and signaling, resulting in the excessive production of extracellular matrix (ECM) proteins. The atypical expression of the 78-kDa glucose-regulated protein (GRP78) on the cell surface may be associated with this pro-fibrotic effect through its interaction with the TGF-β1 activation process. However, there is no current literature demonstrating the role of cell surface GRP78 (csGRP78) in the pathogenesis of diabetic renal diseases. The purpose of my MSc project was to determine the role of csGRP78 in TGF-β1 synthesis and activation and thereby in the progression of DN. We hypothesized that the increased expression of csGRP78 in response to high glucose exposure stimulates TGF-β1 upregulation through intracellular signaling, as well as its activation through interaction with the latent complex, which leads to the expansion of mesangial matrix. / Thesis / Master of Science (MSc) / Diabetic kidney disease affects around 40% of diabetic patients worldwide and is a major health concern. A major feature of the disease is glomerulosclerosis, which is the scarring of glomeruli. The glomeruli filter blood passing through blood vessels in the kidneys to remove waste, which will then be excreted into urine. In diabetic patients, high blood glucose causes the fibrosis of glomeruli and damages the filtration barrier. As a result, a large amount of proteins leak from the blood into the urine. It has been discovered that TGF-β1 is one of the key molecules mediating the generation of scar tissue in the glomerulus. It promotes the growth of mesangial cells, a major type of kidney glomerular cells, and stimulates their production of extracellular matrix proteins. Our results showed that GRP78, a protein that is primarily expressed in the endoplasmic reticulum and assists with protein folding, moves from the inside of cells to the surface in response to a high glucose environment. Here, we found that it facilitated TGF-β1 signaling. Based on our studies, we propose that when GRP78 is at the cell surface, it enables the release of latent TGF-β1, increasing TGF-β1 activity and thus promoting the development of disease.

Diabetic retinopathy (DR) is an incurable, chronic disease that is the leading cause of blindness in working-age adults. A prominent characteristic of DR is the extensive dysfunction within the retina microvasculature. Specialized vascular cells known as pericytes (PCs) are lost or become dysfunctional during disease progression; a thickening of the extracellular matrix (ECM) composing the vascular basement membrane (vBM) and endothelial cell (EC) tight junction disruption are also key features of this disease and contribute to its pathogenesis. PC loss is believed to be a central cue for disease initiation. However, studies inducing PC loss and observing acute changes in the vasculature did not report severe vessel damage or vBM thickening, suggesting that the effects of PC loss occur over a longer period of time. Because the chronic effects of PC loss are more difficult to ascertain, especially in a complex condition such as DR, the mechanisms underlying microvascular defects in DR remain poorly understood. The work presented in this dissertation focuses on pericyte-endothelial cell interactions and their interplay with the ECM/vBM during a variety of physiological and pathological conditions. First, we isolated and functionally validated a primary mouse embryonic PC cell line that we then applied to a co-culture model with ECs to better understand the dynamic interactions between these two critical components of the capillary wall. In the co-culture model, we found that primary PCs promoted EC organization into vessel-like structures and enhanced EC-EC junctions. To complement these in vitro studies, we analyzed animal models and human tissue for the PC-EC interactions and ECM/vBM remodeling under different conditions (physiological and pathological). Moreover, we analyzed microglia and astrocytes to enhance our understanding of the tissue-vessel interface, bolstering our experimental results and facilitating the generation of more hypotheses for future research. Overall, our work suggests that PC-EC interactions in diabetic scenarios play a crucial role in ECM/vBM remodeling; engagement with the ECM/vBM in turn impacted PC behaviors including migration away from the endothelium and induced EC loss of tight junctions, key changes in the onset and progression of DR. / Doctor of Philosophy / Diabetic retinopathy is a group of eye diseases occurring in patients suffering from diabetes and is the leading cause of adult blindness among the working-aged. About one in three people with diabetes over the age of 40 have overt signs of DR. The primary cause for this disease is long-term, high blood sugar levels that damages blood vessels systemically as well as in the eye. Current treatments for DR can prevent the condition from getting worse, but no treatment exists that results in a complete cure. This work described in this dissertation focuses on the interactions between vascular pericytes and endothelial cells, two of the main cell types that compose capillaries (i.e. the smallest blood vessels important for oxygen delivery). The studies presented herein also focus on the response of these cells to the extracellular matrix, a scaffold of proteins that surround pericytes and endothelial cells to stabilize blood vessels. We found that extracellular matrix components dramatically increase as a result of the interactions between pericytes and endothelial cells exposed to diabetic conditions. These changes in the extracellular matrix also had important effects on pericytes and endothelial cells and their engagement with their environment and other cells. Taken together, our work suggests that pericyte-endothelial cell interactions and their crosstalk with the ECM play an important role in blood vessel formation and in the accumulation of microvascular defects that fuel diabetic retinopathy progression.

No / The establishment of a correct diagnosis links care to established guidelines and underpins all subsequent therapeutic activity. Problems can arise when definitions of disease overlap, as is the case with diabetic foot ulceration and pressure ulcers on the foot occurring in people with diabetes. In such cases, clinicians must ensure that patients receive a care bundle that recognises both the wound causation (pressure and shear) and the underlying pathology (diabetic neuropathy, potential foot architecture disruption and ischaemia). All patients with diabetes that have foot ulceration, irrespective of wound aetiology should, therefore, be seen by the multidisciplinary diabetic foot team. Care can then be optimised to include appropriate assessments, including assessment of peripheral perfusion, correct offloading, appropriate diabetic management, and general foot and skin care.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Aside from an indirect effect of PPARgamma activation to reduce insulin resistance and to facilitate adiponectin release, PPARgamma agonist could also exert direct effects on blood vessels. I provided a first line of experimental evidence demonstrating that PPARgamma agonist rosiglitazone up-regulates the endothelin B receptor (ETBR) expression in mouse aortas and attenuates endothelin-1-induced vasoconstriction through an endothelial ET BR-dependent NO-related mechanism. ETBR up-regulation inhibits endothelin-1-induced endothelin A receptor (ETAR)-mediated constriction in aortas and mesenteric resistance arteries, while selective ETBR agonist produces endothelium-dependent relaxations in mesenteric resistance arteries. Chronic treatment with rosiglitazone in vivo or acute exposure to rosiglitazone in vitro up-regulate the ETsR expression without affecting ETAR expression. These results support a significant role of ETBR in contributing to the increased nitric oxide generation upon stimulation with PPARgamma agonist. This study provides additional explanation for how PPARgamma activation improves endothelial function. / Firstly, I demonstrated that adipocyte-derived adiponectin serves as a key link in PPARgamma-mediated amelioration of endothelial dysfunction in diabetes. Results from ex vivo fat explant culture with isolated arteries showed that PPARgamma expression and adiponectin synthesis in adipose tissues correlate with the degree of improvement of endothelium-dependent relaxation in aortas from diabetic db/db mice. PPARgamma agonist rosiglitazone elevates the adiponectin release and restores the impaired endothelium-dependent relaxation ex vivo and in vivo, in arteries from both genetic and diet-induced diabetic mice. The effect of PPARgamma activation on endothelial function that is mediated through the adiponectin- AMP-activated protein kinase (AMPK) cascade is confirmed with the use of selective pharmacological inhibitors and adiponectin -/- or PPARgamma+/- mice. In addition, the benefit of PPARgamma activation in vivo can be transferred by transplanting subcutaneous adipose tissue from rosiglitazone-treated diabetic mouse to control diabetic mouse. I also revealed a direct effect of adiponectin to rescue endothelium-dependent relaxation in diabetic mouse aortas, which involves both AMPK and cyclic AMP-dependent protein kinase signaling pathways to enhance nitric oxide formation accompanied with inhibition of oxidative stress. These novel findings clearly demonstrate that adipocyte-derived adiponectin is prerequisite for PPARgamma-mediated improvement of endothelial function in diabetes, and thus highlight the prospective of subcutaneous adipose tissue as a potentially important intervention target for newly developed PPARgamma agonists in the alleviation of diabetic vasculopathy. / To summarize, the present investigation has provided a few lines of novel mechanistic evidence in support for the positive roles of PPARgamma and PPARdelta activation as potentially therapeutic targets to combat against diabetic vasculopathy. / Type 2 diabetes mellitus and obesity represent a global health problem worldwide. Most diabetics die of cardiovascular and renal causes, thus increasing the urgency in developing effective strategies for improving cardiovascular outcomes, particularly in obesity-related diabetes. Recent evidence highlights the therapeutic potential of peroxisome proliferators activated receptor (PPAR) agonists in improving insulin sensitivity in diabetes. / While agonists of PPARalpha and PPARgamma are clinically used, PPARdelta is the remaining subtype that is yet to be a target for current therapeutic drugs. Little is available in literature about the role of PPARdelta in the regulation of cardiovascular function. The third part of my thesis focused on elucidating cellular mechanisms underlying the beneficial effect of PPARdelta activation in the modulation of endothelial function in diabetes. PPARdelta agonists restore the impaired endothelium-dependent relaxation in high glucose-treated aortas and in aortas from diabetic db/db mice through activation of a cascade involving PPARdelta, phosphatidylinositol 3-kinase, and Akt. PPARdelta activation increases Akt and endothelial nitric oxide synthase and nitric oxide production in endothelial cells. The crucial role of Akt is confirmed by selective pharmacological inhibitors and transient transfection of dominant negative Akt plasmid in these cells. Treatment with PPARdelta agonist GW501516 in vivo augments endothelial function in diabetic db/db and diet-induced obese mice. The specificity of GW501516 for PPARdelta is proven with the loss of its effect against high glucose-induced impairment of endothelium-dependent relaxation in aortas from PPARdelta knockout mice. In addition, oral administration of GW501516 in vivo fails to improve endothelial function in diet-induced obese PPARdelta deficient mice. / Tian, Xiaoyu. / Adviser: Huang Yu. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 132-165). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

研究介紹：炎症與纖維化是糖尿病腎病（DN）的主要特徵。研究發現生長轉化因子TGF-β/Smad信號在糖尿病所致炎症與纖維化中均起重要作用。我們認為TGF-β/Smad信號通路失調是導致糖尿病腎損傷的主要機制，恢復信號通路或有治療價值。為此我們通過以下研究證實：（1）研究Smad7基因在DN中的作用，及評估Smad7基因治療效果；（2）研究miR-29在DN中的作用，及評估miR-29基因治療效果；（3）研究C反應蛋白（CRP）在DN中的作用及機制。 / 研究方法：（1）利用Smad7基因敲除（KO）小鼠建立糖尿病小鼠，並研究Smad7基因在DN的作用，並在链脲佐菌素（STZ）誘導的糖尿病大鼠上利用微泡導入Smad7基因治療觀察其療效；（2）在10週齡db/db小鼠上利用微泡導入可誘導的miR-29b基因，觀察miR-29b在糖尿病腎病中的作用，並用miR-29敲除或高表達細胞株研究其機制；（3）利用CRP轉基因小鼠誘導糖尿病，觀察CRP在DN中的作用，及以高糖和/或CRP刺激腎小管細胞研究CRP的致病機制。 / 研究結果：我們發現（1）糖尿病Smad7 KO小鼠出現更嚴重的腎損傷，包括蛋白尿增加，腎臟炎症及纖維化加重。進一步研究發現Smad7下調所致TGF-β/Smad和NF-kB信號過度活化是導致腎臟炎症及纖維化加重的重要原因。運用基因治療恢復糖尿病大鼠的Smad7水平，發現能夠減輕蛋白尿增加，及抑制TGF-β/Smad引起的纖維化和NF-kB所致炎症反應；（2）我們發現miR-29b在20週齡db/db小鼠比10週齡的顯著降低，並伴隨有蛋白尿加重，腎臟纖維化和炎症反應增加，及TGF-β/Smad，NF-kB，T-bet信號上調，而miR-29b基因治療能減輕蛋白尿，及減輕腎臟纖維化和炎症反應增加，及TGF-β/Smad，NF-kB，T-bet信號上調。體外實驗證實AGEs刺激miR-29敲除細胞株增加纖維化，伴隨有TGF-β/Smad3及炎症因子上調，而刺激高表達細胞株能抑制纖維化，及TGF-β/Smad和炎症因子下調；（3）糖尿病CRP轉基因小鼠出現更嚴重的腎損傷，出現蛋白尿和腎損傷分子-1上升、巨噬細胞和T細胞侵潤、炎症和纖維化增加，並伴有CRP受體（CD32a）上調、TGF-β/Smads及NFκB/p65信號過度活化。體外實驗進一步證實CRP通過其受體CD32a/CD64增加炎症和纖維化。另外證實CRP與高糖有協同作用。 / 結論：TGF-β/Smad信號通路是糖尿病腎病的重要致病機制。糖尿病腎病導致Smad7、miR-29b下調，運用基因治療恢復其表達能減輕糖尿病腎損傷。 / Diabetic nephropathy (DN) is characterized by renal fibrosis and inflammation. Increasing evidence shows that TGF-β/Smad signaling plays a critical role in DN. This thesis tested a hypothesis that TGF-β/Smad signaling may play a central role in diabetic kidney injury and targeting this pathway may represent a novel therapy for DN. The hypothesis was tested in a type-1 model of diabetes induced in Smad7 knockout (KO) or CRP transgene, and the therapeutic potential for DN was examined by overexpressing renal Smad7 or miR-29b in both type-1 or type-2 models of diabetes. / As described in Chapter Three, the protective role and therapeutic potential of Smad7 in diabetic kidney disease was investigated in streptozotocin-induced diabetic model in Smad7 KO mice and in diabetic rats given Smad7 gene transfer using an ultrasound-microbubble-mediated technique. Results showed that Smad7 KO mice developed more severe diabetic kidney injury than wild type (WT) mice as evidenced by a signicant increase in microalbuminuria, renal brosis, and renal inammation, which was mediated by enhanced activation of both TGF-β/Smads and NF-κB signaling pathways. To develop a therapeutic potential for diabetic kidney disease, Smad7 gene was transferred into the kidney, which results in high levels renal Smad7, thereby blocking microalbuminuria, TGF-β/Smad3-mediated renal brosis and NF-κB/p65-driven renal inammation in diabetic rats. / To test a novel hypothesis that TGF-β/Smad3-mediated DN via the Smad3-dependent miR-29, in Chapter Four, the role and mechanisms of miR-29b in DN were examined in vitro in a stable mesangial cell line with overexpression or knockdown of miR-29b and the therapeutic effect of miR-29b on DN was developed by delivering a Dox-inducible miR-29b into 10-week db/db mice. Results showed that addition of AGEs induced a loss of miR-29b with increased fibrosis and inflammation in mesangial cells, which was further enhanced with miR-29b knockdown, but inhibited by overexpressing miR-29b. In db/db mice, reduction of renal miR-29b over the 20 week time was associated with a marked increase in microabluminuria, renal fibrosis and inflammation. Restoring miR-29b resulted in inhibition of kidney injuries by blocking TGF-β/Smad3-mediated renal fibrosis, NF-kB/p65-driven renal inflammation, and importantly, the Th1-dependent immune response, revealing a critical role and therapeutic potential for miR-29b in the pathogenesis of DN. / Finally, diabetic kidney injury was also assessed in under high inflammation conditions in CRP transgenic (Tg) mice. As shown in Chapter Five, CRP Tg mice developed more severe diabetic kidney injury than WT mice, as evidenced by a significant increase in microalbuminuria and kidney injury molecule-1, macrophages and T cells, and upregulation of pro-inflammatory cytokines and extracellular matrix. CRP-mediated DN was associated with upregulation of CRP receptor, CD32a, and over-activation of the TGF-β/Smads and NFκB/p65 signaling pathways. These findings were further confirmed in vitro under high levels of CRP. In addition, CRP was induced by high glucose, which synergistically promoted high glucose-mediated renal inflammation and fibrosis, suggesting a positive feedback-loop between CRP and high glucose under diabetic conditions. / In conclusion, TGF-β/Smads play critical roles in the pathogenesis of DN. Loss of renal Smad7 and miR-29b may be a key mechanism of DN. Thus, over-expression of Smad7 or miR-29b may represent novel therapeutic strategies for diabetic kidney complications. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Haiyong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 202-236). / 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.ii / DECLARATION --- p.vi / ACKNOWLEDGEMENT --- p.vii / PUBLICATIONS --- p.ix / PRESENTATIONS/AWARDS --- p.xi / TABLE OF CONTENTS --- p.xii / LIST OF ABBREVIATIONS --- p.xxii / LIST OF FIGURES/TABLES --- p.xxiv / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- TGF-β superfamily --- p.2 / Chapter 1.2 --- TGF-β/Smad signaling pathway --- p.3 / Chapter 1.2.1 --- TGF-β --- p.3 / Chapter 1.2.1.1 --- TGF-β structure --- p.3 / Chapter 1.2.1.2 --- Activation of latent TGF-β --- p.4 / Chapter 1.2.1.3 --- Latent TGF-β receptors --- p.6 / Chapter 1.2.2 --- TGF-β signaling pathway --- p.7 / Chapter 1.2.2.1 --- Receptors --- p.7 / Chapter 1.2.2.2 --- Smads --- p.10 / Chapter 1.2.2.3 --- Smad-dependent TGF-β signaling pathways --- p.13 / Chapter 1.2.2.4 --- Smad-independent TGF-β signaling pathways --- p.14 / Chapter 1.3 --- Diabetes nephropathy --- p.15 / Chapter 1.3.1 --- Diabetes Mellitus --- p.15 / Chapter 1.3.2 --- Type 1 and type 2 diabetes --- p.16 / Chapter 1.3.3 --- Diabetic complications --- p.16 / Chapter 1.3.4 --- Cellular and molecular mechanisms in diabetic complications --- p.17 / Chapter 1.3.4.1 --- Increased polyol pathway flux --- p.17 / Chapter 1.3.4.2 --- Increased advanced glycation end-products (AGEs) formation --- p.18 / Chapter 1.3.4.3 --- Activation of protein kinase C (PKC) isoforms --- p.20 / Chapter 1.3.4.4 --- Increased hexosamine pathway flux --- p.22 / Chapter 1.3.4.5 --- Increased Reactive Oxygen Species --- p.23 / Chapter 1.3.5 --- Diabetic kidney injuries --- p.24 / Chapter 1.3.5.1 --- Exacerbation of renal structure and function --- p.24 / Chapter 1.3.5.2 --- Fibrosis in diabetic nephropathy --- p.25 / Chapter 1.3.5.3 --- Inflammation in diabetic nephropathy --- p.26 / Chapter 1.4 --- Role of TGF-β/Smad signaling pathway in diabetic nephropathy --- p.28 / Chapter 1.4.1 --- Smad-depedent signaling in diabetic nephropathy --- p.28 / Chapter 1.4.2 --- Cross talk between Smads and other signaling pathways in diabetic nephropathy --- p.30 / Chapter 1.4.3 --- TGF-β/Smads and MicroRNA regulation in diabetic nephropathy --- p.32 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.35 / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Regents and equipment --- p.36 / Chapter 2.1.1.1 --- Reagents and equipment for cell culture --- p.36 / Chapter 2.1.1.2 --- Reagents and equipment for real-time RT-PCR --- p.37 / Chapter 2.1.1.3 --- Reagents and equipment for western blotting --- p.38 / Chapter 2.1.1.4 --- Reagents and equipment for immunohistochemistry --- p.39 / Chapter 2.1.1.5 --- Reagents and equipment for in situ hybridization --- p.40 / Chapter 2.1.1.6 --- Reagents and equipment for plasmid purification --- p.40 / Chapter 2.1.1.7 --- Reagents and equipment for genotyping --- p.41 / Chapter 2.1.1.8 --- Other reagents --- p.41 / Chapter 2.1.2 --- Buffers --- p.42 / Chapter 2.1.2.1 --- Western blotting buffer --- p.42 / Chapter 2.1.2.2 --- Immunohistochemistry buffer --- p.45 / Chapter 2.1.2.3 --- ELISA buffers --- p.47 / Chapter 2.1.2.4 --- In Situ hybridization buffer --- p.48 / Chapter 2.2.2 --- Antibodies --- p.49 / Chapter 2.2.3 --- Primer sequences --- p.49 / Chapter 2.2 --- Methods --- p.56 / Chapter 2.2.1 --- Animal model --- p.56 / Chapter 2.2.1.1 --- Animals --- p.56 / Chapter 2.2.1.2 --- Diabetic animal models --- p.57 / Chapter 2.2.2 --- Sample Collection --- p.59 / Chapter 2.2.2.1 --- Urine collection --- p.59 / Chapter 2.2.2.2 --- Plasma collection --- p.59 / Chapter 2.2.2.3 --- Tissue collection --- p.60 / Chapter 2.2.2.4 --- Paraffin embedding --- p.60 / Chapter 2.2.3 --- Ultrasound-microbubble-mediated gene transfer system --- p.61 / Chapter 2.2.3.1 --- Smad7 gene therapy --- p.61 / Chapter 2.2.3.2 --- miR-29 gene therapy --- p.62 / Chapter 2.2.4 --- Microalbumin and renal function --- p.63 / Chapter 2.2.4.1 --- Microalbuminuria --- p.63 / Chapter 2.2.4.2 --- Creatinine measurement --- p.63 / Chapter 2.2.5 --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.64 / Chapter 2.2.6 --- Histology and immunohistochemistry --- p.64 / Chapter 2.2.6.1 --- Tissue process --- p.64 / Chapter 2.2.6.2 --- Periodic Acid-Schiff Staining (PAS) --- p.64 / Chapter 2.2.6.3. --- Immunohistochemistry (IHC) --- p.65 / Chapter 2.2.6.4 --- In Situ hybridization --- p.66 / Chapter 2.2.6.5 --- Quantitation of histology and IHC --- p.67 / Chapter 2.2.7 --- Cell culture --- p.67 / Chapter 2.2.8 --- Real-time polymerase chain reaction (PCR) --- p.69 / Chapter 2.2.9 --- Western Blotting --- p.70 / Chapter 2.3 --- Statistical analysis --- p.71 / Chapter CHAPTER THREE --- THE PROTECTIVE ROLE OF SMAD7 IN DIABETIC NEPHROPATHY --- p.72 / Chapter 3.1 --- Introduction --- p.73 / Chapter 3.2 --- Materials and methods --- p.74 / Chapter 3.2.1 --- Animal models --- p.74 / Chapter 3.2.2 --- Ultrasound-mediated gene transfer of inducible Smad7 gene-bearing microbubbles into the kidney --- p.74 / Chapter 3.2.3 --- Real-time PCR --- p.75 / Chapter 3.2.4 --- Western blotting --- p.75 / Chapter 3.2.5 --- Microalbuminuria and urinary creatinine analysis --- p.76 / Chapter 3.2.6 --- Histology and immunohistochemistry --- p.76 / Chapter 3.2.7 --- Statistical analysis --- p.77 / Chapter 3.3 --- Results --- p.77 / Chapter 3.3.1 --- Genotyping for Smad7 KO and WT mice --- p.77 / Chapter 3.3.2 --- Disruption of Smad7 enhances diabetic kidney injury --- p.78 / Chapter 3.3.3 --- Disruption of Smad7 enhanced fibrosis in diabetic kidney --- p.80 / Chapter 3.3.3.1 --- Collagen I is enhanced in diabetic Smad7 KO mice --- p.81 / Chapter 3.3.3.2 --- Collagen IV is enhanced in diabetic Smad7 KO mice --- p.82 / Chapter 3.3.3.3 --- Fibronectin is enhanced in diabetic Smad7 KO mice --- p.84 / Chapter 3.3.4 --- Disruption of Smad7 exacerbates inflammation in diabetic kidney --- p.85 / Chapter 3.3.4.1 --- Disruption of Smad7 increases IL-1β in diabetic kidney --- p.85 / Chapter 3.3.4.2 --- Disruption of Smad7 increases TNF-α in diabetic kidney --- p.86 / Chapter 3.3.4.3 --- Disruption of Smad7 Increases MCP-1 in diabetic kidney --- p.87 / Chapter 3.3.4.4 --- Disruption of Smad7 increases ICAM-1 in diabetic kidney --- p.88 / Chapter 3.3.4.5 --- Disruption of Smad7 increases macrophage infiltration in diabetic kidney --- p.90 / Chapter 3.3.5 --- Enhanced activation of TGF-β/Smad3 and NF-κB Signaling is a central mechanism by which disruption of Smad7 promotes diabetic renal fibrosis and inflammation --- p.91 / Chapter 3.3.5.1 --- Smad7 decreases in diabetic kidney --- p.91 / Chapter 3.3.5.2 --- Enhanced activation of TGF-β/Smad3 signaling pathway contributes to fibrosis in diabetic kidney --- p.92 / Chapter 3.3.5.3 --- Enhanced activation of NF-κB/p65 signaling pathway contributes to inflammation in diabetic kidney --- p.93 / Chapter 3.3.6 --- Smad7 transfection rate by gene therapy in diabetic rats --- p.94 / Chapter 3.3.7 --- Restoring Smad7 attenuates kidney injury in diabetic rats --- p.96 / Chapter 3.3.8 --- Restoring Smad7 attenuates renal fibrosis in diabetic rats --- p.98 / Chapter 3.3.8.1 --- Restoring Smad7 attenuates collagen I in diabetic kidney --- p.98 / Chapter 3.3.8.2 --- Restoring Smad7 attenuates collagen IV in diabetic kidney --- p.100 / Chapter 3.3.8.3 --- Restoring Smad7 attenuates collagen III in diabetic kidney --- p.101 / Chapter 3.3.9 --- Restoring Smad7 attenuates renal inflammation in diabetic rats --- p.104 / Chapter 3.3.9.1 --- Restoring Smad7 attenuates IL-1b in diabetic kidney --- p.104 / Chapter 3.3.9.2 --- Restoring Smad7 attenuates TNF-α in diabetic kidney --- p.106 / Chapter 3.3.9.3 --- Restoring Smad7 Attenuates MCP-1 in diabetic kidney --- p.107 / Chapter 3.3.9.4 --- Restoring Smad7 attenuates ICAM-1 in diabetic kidney --- p.109 / Chapter 3.3.9.5 --- Restoring Smad7 attenuates macrophage infiltration in diabetic kidney --- p.111 / Chapter 3.3.10 --- Blockade of activation of TGF-β/Smad3 and NF-κB signaling is a key mechanism by which overexpression of smad7 inhibits diabetic renal injury --- p.113 / Chapter 3.3.10.1 --- Restoring Smad7 inhibits activation of TGF-β/Smad3 signaling --- p.113 / Chapter 3.3.10.2 --- Restoring Smad7 inhibits activation of NF-κB signaling --- p.115 / Chapter 3.3 --- Discussion --- p.117 / Chapter CHAPTER FOUR --- THE PROTECTIVE ROLE OF MICRORNA-29B IN DIABETIC NEPHROPATHY --- p.121 / Chapter 4.1 --- Introduction --- p.122 / Chapter 4.2 --- Materials and methods --- p.123 / Chapter 4.2.1 --- Animal model --- p.123 / Chapter 4.2.2 --- Ultrasound-microbubble-mediated-miR-29 gene transfer --- p.124 / Chapter 4.2.3 --- Real-time polymerase chain reaction (PCR) --- p.124 / Chapter 4.2.4 --- Western Blotting --- p.125 / Chapter 4.2.5 --- Albumin excretion measurement --- p.126 / Chapter 4.2.6 --- ELISA --- p.126 / Chapter 4.2.7 --- Histology and immunohistochemistry --- p.126 / Chapter 4.2.8 --- In Situ hybridization --- p.127 / Chapter 4.2.9 --- Cell culture --- p.128 / Chapter 4.2.10 --- Statistical analysis --- p.129 / Chapter 4.3 --- Results --- p.129 / Chapter 4.3.1 --- Over-expression of miR-29b attenuates, but knockdown of miR-29b enhances fibrosis in vitro --- p.129 / Chapter 4.3.1.1 --- Over-expression of miR-29b attenuates fibrosis --- p.129 / Chapter 4.3.1.2 --- Knockdown of miR-29b enhances fibrosis --- p.132 / Chapter 4.3.2 --- Restoring miR-29b attenuates kidney injury in db/db mice --- p.134 / Chapter 4.3.3 --- Restoring miR-29b attenuates renal fibrosis in db/db mice --- p.139 / Chapter 4.3.3.1 --- Restoring miR-29b attenuates collagen IV in db/db mice --- p.139 / Chapter 4.3.3.2 --- Restoring miR-29b attenuates collagen I in db/db mice --- p.141 / Chapter 4.3.3.3 --- Restoring miR-29b attenuates fibronectin in db/db mice --- p.144 / Chapter 4.3.4 --- Restoring miR-29b inhibits renal fibrosis via TGF-β/Smad3 dependent pathway --- p.146 / Chapter 4.3.5 --- Restoring miR-29b inhibits th1 immune response in diabetic kidney --- p.148 / Chapter 4.3.6 --- Restoring miR-29b inhibits inflammation in diabetic kidney --- p.151 / Chapter 4.4 --- Discussion --- p.154 / Chapter 4.5 --- Conclusion --- p.161 / Chapter CHAPTER FIVE --- THE PATHOGENIC ROLE OF C-REACTIVE PROTEIN IN DIABETIC NEPHROPATHY --- p.162 / Chapter 5.1 --- Introduction --- p.163 / Chapter 5.2 --- Materials and methods --- p.164 / Chapter 5.2.1 --- Mouse model of STZ induced diabetes --- p.164 / Chapter 5.2.2 --- Measurement of blood glucose, urinary albumin excretion, and creatinine clearance --- p.165 / Chapter 5.2.3 --- Histology and immunohistochemistry --- p.165 / Chapter 5.2.4 --- Cell culture --- p.166 / Chapter 5.2.5 --- Real-time PCR --- p.166 / Chapter 5.2.6 --- Western blotting --- p.167 / Chapter 5.2.7 --- Statistical analyses --- p.168 / Chapter 5.3 --- Results --- p.168 / Chapter 5.3.1 --- Diabetic renal injury is exacerbated in CRP Tg mice --- p.168 / Chapter 5.3.2 --- Renal inflammation is exacerbated in diabetic CRP Tg mice --- p.172 / Chapter 5.3.2.1 --- F4/80+ macrophage infiltration is enhanced in diabetic CRP Tg mice --- p.172 / Chapter 5.3.2.2 --- CD3+ T cell infiltration is enhanced in diabetic CRP Tg mice --- p.173 / Chapter 5.3.2.3 --- TNF-α expression is enhanced in diabetic CRP Tg mice --- p.173 / Chapter 5.3.2.4 --- IL-1β expression is enhanced in diabetic CRP Tg mice --- p.174 / Chapter 5.3.3 --- Renal fibrosis is enhanced in diabetic CRP Tg mice --- p.175 / Chapter 5.3.3.1 --- Collagen I is enhanced in Diabetic CRP Tg mice --- p.175 / Chapter 5.3.3.2 --- Collagen IV is enhanced in diabetic CRP Tg mice --- p.176 / Chapter 5.3.4 --- Enhanced CRP signaling and activation of NF-κB and TGF-β/Smad3 signaling are key mechanism by which CRP promotes diabetic renal injury --- p.177 / Chapter 5.3.4.1 --- Enhanced CRP signaling via upregulation of CD32a expression --- p.177 / Chapter 5.3.4.2 --- enhanced activation of NF-κB signaling is key mechanism by which CRP promotes renal inflammation --- p.179 / Chapter 5.3.4.3 --- Enhanced activation of TGF-β/Smad3 signaling is key mechanism by which CRP promotes renal inflammation --- p.181 / Chapter 5.4 --- Discussion --- p.194 / Chapter 5.5 --- Conclusion --- p.197 / Chapter CHAPTER SIX --- SUMMARY AND CONCLUSION --- p.198 / REFERENCES --- p.202

Ho, Wing-Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 115-131). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.v / 摘要 --- p.ix / Publications --- p.xii / Table of Contents --- p.xiv / Chapter 1. --- General Introduction / Chapter 1.1. --- Diabetes Mellitus (DM) and Diabetic Nephropathy --- p.1 / Chapter 1.1.1. --- "Prevalence, Diagnosis and Classification of DM" --- p.1 / Chapter 1.1.2. --- Type 2 DM and its Complications: Diabetic Nephropathy --- p.5 / Chapter 1.1.3. --- Diagnosis and Impacts of Diabetic Nephropathy --- p.7 / Chapter 1.1.4. --- Current Treatment of Type 2 DM and Diabetic Nephropathy --- p.8 / Chapter 1.2. --- Cytokines and Chemokines --- p.9 / Chapter 1.2.1. --- Types and Properties --- p.9 / Chapter 1.2.2. --- Cytokines and chemokines in Type 2 DM and Diabetic Nephropathy --- p.13 / Chapter 1.3. --- T Lymphocyte Costimulatory Molecules --- p.15 / Chapter 1.3.1. --- Types and Properties --- p.15 / Chapter 1.3.2. --- T Lymphocyte Costimulatory Molecules in Type 2 DM and Diabetic Nephropathy --- p.16 / Chapter 1.4. --- Adhesion Molecules --- p.18 / Chapter 1.4.1. --- Types and Properties --- p.18 / Chapter 1.4.2. --- Adhesion Molecules in Type 2 DM and Diabetic Nephropathy --- p.20 / Chapter 1.5. --- Intracellular Signaling Pathways --- p.21 / Chapter 1.5.1. --- Types and Properties --- p.21 / Chapter 1.5.2. --- Intracellular Signaling Pathways in Type 2 DM and Diabetic Nephropathy --- p.23 / Chapter 1.6. --- Objectives of Our Study --- p.24 / Chapter 2. --- Materials and Methods / Chapter 2.1. --- Materials --- p.26 / Chapter 2.1.1. --- "Patients, Control Subjects and Blood Samples" --- p.26 / Chapter 2.1.2. --- Cell Line --- p.27 / Chapter 2.1.3. --- "Cell Culture Media, Buffers and Other Reagents" --- p.28 / Chapter 2.1.4. --- "Recombinant Human Cytokines, Inhibitors and Other Stimulators" --- p.30 / Chapter 2.1.5. --- Reagents and Buffers for Flow Cytometric Analysis --- p.31 / Chapter 2.1.5.1. --- Cytometric Bead Array (CBA) of Cytokines and Chemokines --- p.33 / Chapter 2.1.5.2. --- Multiplex Fluorescent Bead Immunoassay (FBI) of Soluble Adhesion Molecules --- p.33 / Chapter 2.1.5.3. --- Phosphorylation State Analysis of Signaling Molecules --- p.34 / Chapter 2.1.5.4. --- Immunofluorescent Staining of Cell Surface Molecules --- p.36 / Chapter 2.1.6. --- Reagents and Buffers for Protein Array Analysis --- p.37 / Chapter 2.1.7. --- "Reagents and Buffers for 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenylytetrazolium Bromide (MTT) Assay" --- p.37 / Chapter 2.1.8. --- Reagents for Human Enzyme-Linked Immunosorbent Assay (ELISA) --- p.37 / Chapter 2.2. --- Methods --- p.38 / Chapter 2.2.1. --- Whole Blood Culture Experiments --- p.38 / Chapter 2.2.2. --- "Collection of Serum and Plasma, and Purification of PBMC from EDTA-Blood" --- p.39 / Chapter 2.2.3. --- HK-2 Cell Cultures --- p.39 / Chapter 2.2.4. --- HK-2 Cell Treatments --- p.40 / Chapter 2.2.5. --- Flow Cytometric Analysis --- p.41 / Chapter 2.2.5.1. --- CBA of Cytokines and Chemokines --- p.41 / Chapter 2.2.5.2. --- Multiplex FBI of Soluble Adhesion Molecules --- p.41 / Chapter 2.2.5.3. --- Phosphorylation State Analysis of Signaling Molecules --- p.42 / Chapter 2.2.5.4. --- Immunofluorescent Staining of Cell Surface Molecules --- p.43 / Chapter 2.2.6. --- Protein Array Analysis --- p.44 / Chapter 2.2.7. --- MTT Assay --- p.44 / Chapter 2.2.8. --- ELISA --- p.45 / Chapter 2.2.9. --- Statistical Analysis --- p.46 / Chapter 3. --- "Clinical Study on the Expressions of Cytokines, Chemokines, Co-stimulatory Molecules, Phosphorylated Signaling Molecules in Patients with Diabetic Nephropathy" / Chapter 3.1. --- Introduction --- p.47 / Chapter 3.2. --- Results --- p.49 / Chapter 3.2.1. --- Demographic Data of Participants --- p.49 / Chapter 3.2.2. --- Expression Profile in Plasma of Patients --- p.49 / Chapter 3.2.2.1. --- Cytokines and Chemokines --- p.49 / Chapter 3.2.2.2. --- Soluble Costimulatory Molecules --- p.55 / Chapter 3.2.2.3. --- Soluble Adhesion Molecules --- p.55 / Chapter 3.2.2.4. --- "Correlations between Plasma Concentrations of Cytokines, Chemokines, soluble Costimulatory Molecules and soluble Adhesion Molecules and UACR in Patients" --- p.60 / Chapter 3.2.3. --- Effects ofTNF-α and IL-18 on the ex vivo Production from Whole Blood of Patients --- p.65 / Chapter 3.2.3.1. --- Ex vivo Production of Cytokines and Chemokines --- p.65 / Chapter 3.2.3.2. --- Ex vivo Production of Soluble Costimulatory Molecules --- p.70 / Chapter 3.2.4. --- "Expression of Phosphorylated p38 MAPK, JNK and ERK in PBMC of Patients" --- p.73 / Chapter 3.3. --- Discussion --- p.77 / Chapter 3.3.1. --- "Cytokines, Chemokines and Diabetic Nephropathy" --- p.77 / Chapter 3.3.2. --- Soluble Costimulatory Molecules and Diabetic Nephropathy --- p.80 / Chapter 3.3.3. --- Soluble Adhesion Molecules and Diabetic Nephropathy --- p.83 / Chapter 3.3.4. --- Intracellular Signaling and Diabetic Nephropathy --- p.87 / Chapter 4. --- In vitro Study on the Signal Transduction Mechanism Regulating the Expression of CCL2 and Cell Surface Adhesion Molecules in Tumour Necrosis Factor (TNF)-α-Stimulated HK-2 Cells / Chapter 4.1. --- Introduction --- p.90 / Chapter 4.2. --- Results --- p.93 / Chapter 4.2.1. --- Expression Profile of Cytokines and Chemokines of TNF-α-activated HK-2 Cells --- p.93 / Chapter 4.2.2. --- "TNF-α Upregulated CCL2, ICAM-1 and VCAM-1 Expression in HK-2 Cells" --- p.95 / Chapter 4.2.3. --- "TNF-α Activated the p38 MAPK, JNK and ERK Signaling Pathways in HK-2 Cells" --- p.96 / Chapter 4.2.4. --- Cytotoxicity of MAPK Inhibitors --- p.96 / Chapter 4.2.5. --- "Effects of p38 MAPK, JNK and ERK Inhibitors on TNF-α-induced Expressions of CCL2, ICAM-1 and VCAM-1" --- p.100 / Chapter 4.3. --- Discussion --- p.102 / Chapter 5. --- Conclusion and Future Prospects / Chapter 5.1. --- Conclusion --- p.107 / Chapter 5.2. --- Future Prospects --- p.111 / References --- p.115