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| Project Details |
| Funding Scheme : | General Research Fund | ||||||||||||
| Project Number : | 769410 | ||||||||||||
| Project Title(English) : | Obesity, adipose tissue inflammation and atherosclerosis: role of JNK activation | ||||||||||||
| Project Title(Chinese) : | 應激活化蛋白激酶在介導脂肪炎症所引起之血管功能障礙中的作用 | ||||||||||||
| Principal Investigator(English) : | Prof Lam, Karen Siu Ling | ||||||||||||
| Principal Investigator(Chinese) : | |||||||||||||
| Department : | Department of Medicine | ||||||||||||
| Institution : | The University of Hong Kong | ||||||||||||
| E-mail Address : | ksllam@hku.hk | ||||||||||||
| Tel : | 2255 4783 | ||||||||||||
| Co - Investigator(s) : |
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| Panel : | Biology & Medicine | ||||||||||||
| Subject Area : | Medicine, Dentistry & Health | ||||||||||||
| Exercise Year : | 2010 / 11 | ||||||||||||
| Fund Approved : | 1,300,000 | ||||||||||||
| Project Status : | Completed | ||||||||||||
| Completion Date : | 31-12-2014 | ||||||||||||
| Project Objectives : |
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| Abstract as per original application (English/Chinese): |
The obesity epidemic, seen in many parts of the world including China, is associated with increased death and suffering due to the atherosclerotic diseases, coronary heart disease and stroke. Inflammation of the fat tissues may be one mechanism whereby obesity leads to enhanced atherosclerosis. Recent research findings suggest that obesity-related nutrition overload and metabolic stress in fat cells lead to excess burdens to the cellular machineries, in turn resulting in the activation of various inflammatory pathways, including the c-Jun N-terminal kinase (JNK) pathway. We have shown that JNK activation in the fat cells enhances the production of various pro-inflammatory cell products, or cytokines, and the infiltration of fat tissues by inflammatory cells called macrophages. These cytokines, also produced by the macrophages, can accelerate the development of atherosclerosis by promoting insulin resistance and its related cardiovascular risk factors including hyperglycaemia, hypertension and hypertension, by increasing the macrophage infiltration of the vascular tress, and by reducing the production of adiponectin, an insulin-sensitizing, anti-inflammatory and anti-atherogenic hormone. It is now recognized that atherosclerosis is an inflammatory disease characterized by the infiltration of inflammatory cells and the presence of foam cells or cholesterol-filled macrophages. We have recently shown that adiponectin can increase circulating endothelial progenitor cells (EPC) in obese diabetic db/db mice which have impaired EPC-mediated vascular repair. Reduced circulating EPC number is also found in humans with cardiovascular disease and/or various cardiovascular risk factors including diabetes and hyperlipidaemia, and predicts cardiovascular outcome, suggesting the importance of EPC in vascular integrity and protection from atherosclerotic diseases. EPC survival, differentiation and function can be adversely affected by inflammatory mediators. JNK activation, by impairing vascular response to insulin, can also reduce the production of nitric oxide, a molecule of crucial importance in EPC-mediated vascular repair. The above findings have prompted us to hypothesize that JNK activation in fat tissues may play a central role in linking obesity with atherosclerosis. We will test this hypothesis in the db/db mice and the hypercholesterolaemic, atherosclerosis-prone apo E deficient mice, by selectively blocking JNK activation in their fat tissues using transgenic technologies. We will investigate the effects on vascular repair and whether they are consequent to changes in EPC mobilization or function. The development of atherosclerotic lesions will also be assessed. In addition to improving our understanding of how obesity can lead to atherosclerotic diseases, this project may potentially lead to a new therapeutic approach to obesity-related atherosclerosis. 在現今世界，包括中國在內，肥胖症正日益流行，此與動脈粥樣硬化、冠心病和中風導致的死亡和病痛的增加有密切相關。最近的研究結果表明，與肥胖有關的營養過剩和脂肪細胞代謝會激使細胞機器負擔過重，從而激活了各種炎症反應的信號通路，包括JNK路徑。我們已經證實，在脂肪細胞內，JNK的激活導致了各種炎症免疫蛋白因子或細胞因子的產生，以及脂肪組織內巨噬細胞的浸潤。這些由巨噬細胞產生的細胞因子，通過進一步增加巨噬細胞浸潤和減少脂聯素（一種有胰島素增敏，抗炎和抗動脈粥樣硬化作用的激素）的生產，從而促進胰島素抵抗以及其相關的心血管疾病的危險因素，包括高血糖、高血壓和高血脂的發生，進而加速了動脈粥樣硬化的發展。現在我們已經認識到，動脈粥樣硬化實際上是一種炎症性疾病，它是以炎性細胞浸潤和泡沫細胞或膽固醇填充的巨噬細胞的存在為特點。最近，我們的實驗證實，脂聯素可以增加db/db肥胖糖尿病小鼠血循環中的內皮祖細胞（EPC）的數量，從而改善肥胖db/db小鼠本身的EPC介導的血管內皮的修復機能。同時，我們已經注意到，在一些患有心血管疾病和/或有各種心血管疾病危險因素（包括糖尿病和高血糖狀態）的人群，在外周血循環中也存在EPC減少的現象，而這種循環中EPC的減少可以預測心血管疾病的結果。這就表明，循環中的EPC在維持血管的完整以及粥樣硬化的防護方面有很重要的作用。炎症介質對EPC的生存，分化和功能有著廣泛的影響。JNK的活化導致血管對胰島素的反應受損，同時還可以減少一氧化氮的產生，而一氧化氮對EPC介導的血管修復至關重要。上述研究結果促使我們提出這樣的假設：在脂肪組織中JNK的活化可能在肥胖導致動脈粥樣硬化的過程中起著關鍵的作用。為了進一步的探索以上的假說，我們將在db/db小鼠和高膽固醇血合併動脈粥樣硬化的載脂蛋白E基因缺陷小鼠中，應用轉基因技術，有選擇性的阻斷脂肪組織中JNK的激活，進一步研究這種JNK活性的阻斷對血管修復的影響，以及是否因此而轉變EPC動員或功能。同時，我們還將評估動脈粥樣硬化病變的發展。這個項目，除了可以幫助我們進一步了解肥胖是如何導致動脈粥樣硬化性疾病的，同時也提出了一個潛在的針對肥胖有關的動脈粥樣硬化的治療靶向。 |
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| Realisation of objectives: | For Objective 1, while cross-breeding ApoE-/- mice with transgenic mice with adipose-specific expression of a dominant negative form of JNK (dnJNK) to generate ApoE-/-/dnJNK (ADJ) mice, we first characterized whether diet-induced obesity and hypercholesterolaemia per se in wildtype (WT) mice could lead to impaired re-endothelialization, using a carotid artery injury model. HFHC diet treatment for 8 weeks significantly enhanced the body weight and % fat without affecting fasting and feeding blood glucose. However, these obese mice exhibited glucose intolerance compared to the lean control as evidenced by glucose intolerance test (GTT). Interestingly, these mice revealed no significant change in reendothelialization upon vascular injury compared with the lean control mice. Both lean and obese mice attained approximately 40% of vascular reendothelialization 3 days after the wire-induced injury in the carotid arteries (see appendices at end of report). These data suggest that obesity and hypercholesterolaemia per se do not result in vascular reendothlialization upon vascular injury. In view of these findings, we concluded that further investigating the role of tissue-specific deletion of JNK on vascular repair in ADJ mice and ApoE-/- mice with diet-induced obesity would not be justified. On the other hand we continued to address the remaining part of Objective 1 and investigated whether adipose-specific inactivation of JNK could protect against atherosclerosis development in hypercholesterolaemic mice on HFHC diet. Our subsequent studies in the ADJ mice showed that selective adipose tissue suppression of JNK activity could indeed protect against atherosclerosis. For Objective 2, to confirmatively demonstrate a long-distance communication between inflamed adipose tissue and the vasculature, we generated an atherogenic mouse model with the locally inflamed adipose tissue residing in the periphery, by transplanting epidermal white adipose tissue (eWAT) isolated from obese donor mice (WT or dn-JNK) into ApoE-/- recipients subcutaneously. We had modified the initially proposed protocol by transplanting the fat from dn-JNK and WT mice fed on HFHC diet, instead of standard diet-fed mice, for 12 weeks, to speed up the development of the atherosclerosis in the recipients. Compared to sham-operated mice, recipient ApoE-/- mice which received eWAT graft from obese WT mice (trans-WT) demonstrated increased atherosclerotic changes and systemic inflammation as evidenced by elevated serum levels of soluble tumour necrosis factor receptor 2 (sTNFR2) and adipocyte fatty acid binding protein (A-FABP). eWAT graft from dnJNK donors displayed significantly reduced inflammation compared to that from WT donors, even after 4 weeks of transplantation. The transplantation of eWAT from dnJNK donors largely reversed the accelerated atherogenesis observed in ApoE-/- recipients of eWAT from WT donors. Thus we have successfully established the visceral fat transplant model and demonstrated the role of JNK in the cross-talk between visceral fat inflammation and atherosclerosis. Because we previously found that A-FABP interacts with JNK via a positive feedback loop to modulate inflammatory responses (Hui X et al, J Biol Chem 2010), we proceeded beyond our original proposal to investigate the effect of continuous infusion of A-FABP into Apo-E-/- recipients of dnJNK donors, to levels comparable to those in recipients of obese wild-type donors. This additional study had provided further mechanistic insight on the JNK-mediated cross-talk between visceral fat and the vasculature. | ||||||||||||
| Summary of objectives addressed: |
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| Research Outcome | |||||||||||||
| Major findings and research outcome: | After 10 weeks of high-fat-high-cholesterol diet treatment, ApoE deficient mice (ApoE-/-) with adipose-specific JNK inactivation, ApoE-/-/dnJNK (ADJ), had significantly reduced atherosclerotic plaques, macrophage accumulation, and smooth muscle cell proliferation in the aortic root region when compared with ApoE-/- mice. A reduced macrophage infiltration was also observed in the epididymal white adipose tissue (eWAT) in ADJ mice, as shown by histology, and the expression levels of pro-inflammatory markers including macrophage chemoattractant protein 1 (MCP-1), tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β), and adipocyte fatty acid binding protein (A-FABP) were significantly lower in eWAT in ADJ mice than in ApoE-/- mice. There was a significant reduction in systemic inflammation in ADJ mice, as evidenced by reduced levels of serum MCP-1 and A-FABP. These significant reductions in visceral fat and systemic inflammation in the ADJ mice, conferred by adipose-specific JNK inactivation, were not associated with significant changes in lipid or glucose metabolism, but were sufficient to provide protection against atherosclerosis in these ApoE deficient mice. Using the visceral fat transplant model, we found that lean ApoE-/- mice that received transplantation of visceral fat from obese wild-type donor mice for 4 weeks showed exacerbated systemic inflammation and atherosclerotic plaque formation. Conversely, lean ApoE-/- recipients carrying visceral fat graft from obese dnJNK donors were protected against increased systemic inflammation, as reflected by circulating levels of pro-inflammatory cytokines including adipocyte fatty acid binding protein (A-FABP), and accelerated atherogenesis. The beneficial effects of adipose-specific JNK inactivation on atherogenesis in ApoE-/- recipients could be significantly abrogated by continuous infusion of recombinant A-FABP to levels comparable to those in recipients of obese wild-type mice. We concluded therefore that enhanced atherosclerosis in obesity can be attributed, at least in part, to a distant cross-talk between visceral fat and the vasculature, mediated by the release of pro-inflammatory adipokines, such as A-FABP, from the inflamed visceral adipose tissue with JNK activation. The above data have been included in a manuscript submitted to Circulation and several abstracts presented at international meetings. | ||||||||||||
| Potential for further development of the research and the proposed course of action: |
A recent publication (Cao H et al, Cell Metabolism 2013) has reported that, in mice, recombinant A-FABP can stimulate hepatic gluconeogenesis whereas treatment with a neutralizing antibody for 2 weeks reduces hepatic glucose production. It should be noted that monoclonal antibodies directed against therapeutic target molecules or receptors are increasingly used in targeted therapy for treating various diseases, including cancers, immunological diseases and severe hypercholesterolaemia. Our findings in the current project would suggest that monoclonal antibodies against A-FABP may be explored for the prevention and treatment of atherosclerotic diseases, such as coronary artery diseases and stroke. On the other hand, we and others have found that obesity (Zhang X et al, Diabetes 2008) and various obesity-related disorders, including diabetes (Chen C et al, Diabetes Care 2011), non-alcoholic steatohepatitis (Dushay J et al,Gastroenterology 2010) and atherosclerosis (Chow WS et al, ATVB 2013), are associated with increased levels of fibroblastic growth factor 21 (FGF21), a metabolic hormone being actively investigated in preclinical and clinical studies for the treatment of diabetes and hyperlipidaemia (Woo YC et al, Clin Endocrinol 2013). The possible presence of FGF21 resistance was demonstrated in obese mice in which impaired FGF21 signaling consequent to JNK-mediated TNF-α activation in the inflamed adipose tissue was observed (Diaz-Defin J et al, Endocrinol 2012). Our ADJ mice can potentially be employed to investigate whether and how adipose tissue FGF21 resistance is involved in the pathogenesis of obesity-related atherosclerotic diseases. | ||||||||||||
| Layman's Summary of Completion Report: | Obesity is associated with increased risk of atherosclerotic diseases, such as heart disease and stroke. In this study, we have demonstrated the importance of fat tissue inflammation in the development of atherosclerosis. It has been observed that individuals with central obesity or excessive fat in their abdomen (so-called visceral fat), have a greater risk of dying from atherosclerotic diseases, than those who cumulate fat tissue mainly under the skin. In this study, we have identified an underlying mechanism for this observation. We have demonstrated that the inflamed visceral fat from obese mice, when transplanted under the skin into lean mice which are prone to atherosclerosis, can accelerate the development of atherosclerosis in these lean mice. This accelerated atherosclerosis can be blocked if the visceral fat from the donor mice is genetically suppressed. Our studies also demonstrate that the distant inter-talk between visceral fat and the blood vessels is mediated by the increased release of inflammatory factors from the visceral fat, such as adipocyte fatty acid binding protein. Therefore, strategies which block the action of these inflammatory factors can potentially be explored for the prevention of coronary artery diseases and stroke, common causes of death in our aging population. | ||||||||||||
| Research Output | |||||||||||||
| Peer-reviewed journal publication(s) arising directly from this research project : (* denotes the corresponding author) |
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| Recognized international conference(s) in which paper(s) related to this research project was/were delivered : |
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| Other impact (e.g. award of patents or prizes, collaboration with other research institutions, technology transfer, etc.): |
Poster entitled 'Selective inactivation of c-jun NH2-terminal kinase in adipose tissue alleviates atherosclerosis in obese ApoE-deficient mice' was awarded Session Best Poster Award at 19th Research Postgraduate Symposium, Faculty of Medicine, HKU, 10-11 Dec 2014, Hong Kong | ||||||||||||
| SCREEN ID: SCRRM00542 |