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| Project Details |
| Funding Scheme : | General Research Fund | |||||||||||||||
| Project Number : | 763312 | |||||||||||||||
| Project Title(English) : | Does an elevated lipocalin-2 concentration in plasma lead to endothelial dysfunction and hypertension? | |||||||||||||||
| Project Title(Chinese) : | 血漿中載脂蛋白-2濃度升高會否導致血管內皮功能障礙和高血壓？ | |||||||||||||||
| Principal Investigator(English) : | Prof Cheung, Bernard Man Yung | |||||||||||||||
| Principal Investigator(Chinese) : | 張文勇 | |||||||||||||||
| Department : | Department of Medicine | |||||||||||||||
| Institution : | The University of Hong Kong | |||||||||||||||
| E-mail Address : | mycheung@hku.hk | |||||||||||||||
| Tel : | 22554347 | |||||||||||||||
| Co - Investigator(s) : |
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| Panel : | Biology & Medicine | |||||||||||||||
| Subject Area : | Medicine, Dentistry & Health | |||||||||||||||
| Exercise Year : | 2012 / 13 | |||||||||||||||
| Fund Approved : | 1,158,894 | |||||||||||||||
| Project Status : | Completed | |||||||||||||||
| Completion Date : | 30-9-2015 | |||||||||||||||
| Project Objectives : |
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| Abstract as per original application (English/Chinese): |
Hypertension and type 2 diabetes mellitus are strongly related to obesity in Hong Kong Chinese. Obesity elevates blood pressure through multiple mechanisms. Lipocalin-2 (also known as neutrophil gelatinase-associated lipocalin) is secreted by fat cells. It is known to be associated with obesity and insulin resistance, but our recent findings in mouse and man suggest that it may also play an important role in hypertension. Mice deficient in it does not develop endothelial dysfunction and hypertension when fed on a high fat diet. When these mice receive lipocalin-2 replacement, they develop endothelial dysfunction. In man, plasma lipocalin-2 level increases with obesity and hypertension. Variants in the gene encoding lipocalin-2 are associated with raised plasma level of lipocalin-2 and hypertension. We postulate that lipocalin-2 adversely affects the relaxation of blood vessels and causes hypertension. We propose to give lipocalin-2 to lipocalin-2 knockout mice for 8 weeks and observe the changes in blood pressure and endothelial function. We shall use two models of obesity (diet-induced and genetic); lipocalin-2 knockout mouse fed with a high fat diet, and a double knockout mouse deficient in both leptin receptor and lipocalin-2 on normal diet. In 800 people who have had measurements of flow-mediated dilatation of the brachial artery to assess endothelial function, we shall measure their plasma lipocalin-2 level. We anticipate that the plasma level of lipocalin-2 will be significantly related to endothelial function in man, and in mice, 8-week administration of lipocalin-2 will lead to endothelial dysfunction and elevation in blood pressure. In over 1900 participants of the Hong Kong Cardiovascular Risk Factor Prevalence cohort, we will also investigate whether or not serum lipocalin-2 level can predict the long-term development of hypertension. This project will provide important insights into one of the mechanisms by which obesity can lead to hypertension, and may lead to the development of new classes of therapeutic agents. 在香港的華人，肥胖與高血壓和2型糖尿病是有密切關係的。肥胖通過多種機制提升血壓。載脂蛋白-2（也被稱為中性粒細胞明膠酶相關脂質運載蛋白） 是由脂肪細胞分泌的。我們知道它與肥胖和胰島素抵抗有相關性，但最近我們在小鼠和人的研究結果發現，它可能在高血壓也發揮了重要的角色。缺乏它的小鼠當餵以高脂肪的飲食時不會發展血管內皮功能障礙和高血壓。當這些小鼠接受載脂蛋白-2補充時，它們便開始患上內皮功能障礙。人類的血漿載脂蛋白-2水平會跟隨肥胖和高血壓而提高。載脂蛋白-2基因編碼的變異與血漿載脂蛋白-2水平以及高血壓相關。我們假設，載脂蛋白-2對血管的鬆弛產生不利的影響和導致高血壓。我們計劃給予載脂蛋白-2基因敲除小鼠8週的載脂蛋白-2，觀察血壓和血管內皮功能的變化。我們將使用兩種肥胖的模型（飲食引起的和遺傳性）: 餵以高脂肪飲食的載脂蛋白-2基因敲除小鼠，以及正常飲食的缺乏瘦素受體和載脂蛋白-2雙基因敲除小鼠。在800名曾經以肱動脈血流介導的擴張來評估血管內皮功能的人，我們將測量他們的血漿載脂蛋白-2的水平。我們預期血管內皮功能和血漿載脂蛋白-2水平將顯著相關，也預期載脂蛋白-2輸入小鼠體內8週會導致血管內皮功能障礙和血壓升高。在超過1900名香港心血管疾病風險因素調查的參與者中，我們也將探討血漿載脂蛋白-2水平是否可以預測長期高血壓的發展。此項目將提供關於肥胖導致高血壓機制其中之一的重要見解，並可能導致新類型治療藥物的發展。 |
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| Realisation of objectives: | Animal studies Amelioration of vascular inflammation, endothelial dysfunction and hypertension in lipocalin-2 knockout mice During the first year of the project, we made the unexpected and important discovery that instead of the native form, it is the deamidated form of lipocalin-2 that causes vascular inflammation, endothelial dysfunction and hypertension in mice fed on a high fat diet. We found that administration of lipocalin-2 in combination with non-esterified free fatty acids, especially linoleic acid (18:2n-6), caused endothelial dysfunction and vascular inflammation in lipocalin-2 knockout mice. Linoleic acid treatment facilitated the deamidation of lipocalin-2, reduced its interactions with matrix metalloproteinase 9 (MMP9), and increased the circulating level as well as the accumulation of this adipokine in the arterial wall. A human lipocalin-2 mutant with the cysteine 87 residue replaced by alanine (C87A) evoked endothelial dysfunction in the absence of combined treatment with linoleic acid. C87A contains a reduced amount of polyamines and exhibits reduced capacity to form heterodimeric complexes with MMP9. Blockage of the latter formation with a MMP9 inhibitor promoted the stabilization and deamidation of lipocalin-2. These results demonstrated that activation of lipocalin-2 by deamidation plays a causal role in causing endothelial dysfunction under conditions of high non-esterified fatty acids intake or dietary obesity. We monitored the blood pressure of lipocalin-2 knockout mice and wild type mice throughout 24 hours using implanted sensors and radiotelemetry. Firstly, we found that lipocalin-2 knockout mice had significantly lower 24-hour average blood pressure compared to wild-type controls (122.4±15.6/108.7±16.2 mmHg vs. 142±16.3/123.4±15.6 mmHg, p<0.01). Moreover, the diurnal variation in blood pressure was blunted in wild type mice on high fat diet but maintained in lipocalin-2 knockout mice despite the same high fat diet. Thus, we have fulfilled the first objective of the grant proposal, namely, to demonstrate that lipocalin-2, in particular, its deamidated from, acts together with a high fat diet to cause accumulation of lipocalin-2 in the arterial wall, provoking inflammation and endothelial dysfunction. Mice deficient in lipocalin-2 are resistant to these harmful effects and the elevation of blood pressure caused by high fat diet. Beyond the objectives of the RGC proposal, we are delighted to report that we have carried out further experiments to understand the mechanism whereby lipocalin-2 causes atherosclerosis and to demonstrate the therapeutic potential of suppressing lipocalin-2. We discovered that lipocalin-2 caused vascular inflammation through neutrophil activation. Neutrophils derived from lipocalin-2 deficient mice could not form neutrophil extracellular trap (NET) upon stimulation by linoleic acid. Replacing with lipocalin-2 and adding linoleic acid restored NET formation. Our results suggested that lipocalin-2 plays an indispensable role in the development of atherosclerosis by promoting neutrophil activation and chronic vascular inflammation. In double knockout mice deficient in apolipoprotein E as well as lipocalin-2, atherosclerosis did not develop even when the mice were fed a high fat diet. In another model of hypertension, in which hypertension in mice was induced by the infusion of angiotensin II or the combination of aldosterone, uninephrectomy and 1% saline in drinking water (ANS), knocking out lipocalin-2 reduced mineralocorticoid receptor expression and prevented the development of hypertension and fibrotic changes in the heart and kidneys. Human studies Lipocalin-2 and hypertension The plasma concentration of lipocalin-2 had been measured by immunoassay in 1925 subjects of the Hong Kong Cardiovascular Risk Factor Prevalence Study (CRISPS) using previous grants, including RGC grant HKU780210M. The results showed that plasma lipocalin-2 level was higher in men than in women (median [IQR] 37.7 [30.5-47.9] vs.31.6 [25.4-40.4], p<0.001). It was significantly related to age (r=0.15, p<0.001) and systolic blood pressure (r=0.15, p<0.001). In women, it was also significantly related to waist circumference (r=0.16, p<0.001), BMI (r=0.09, p=0.004), diastolic blood pressure (r=0.14, p<0.001) and fasting plasma glucose (r=0.089, p=0.004). The funding from the current proposal allowed us to study the relationship between plasma lipocalin-2 at baseline and the development of hypertension. Over a median follow-up of 5.4 years, 239 subjects developed hypertension among 1116 subjects that were normotensive at baseline. In multiple logistic regression analysis, the baseline level of lipocalin-2 in plasma was a significant independent predictor of new-onset hypertension (p=0.017). We concluded that plasma lipocalin-2 concentration is related to systolic blood pressure, and is higher in men and in people with hypertension or predisposed to developing hypertension. Lipocalin-2 and endothelial function Endothelial function was not assessed in CRISPS. Therefore, to study the relationship between lipocalin-2 and endothelial function, we measured plasma lipocalin-2 concentration in the stored blood samples of subjects who had measurements of brachial artery flow-mediated dilatation (FMD) in studies of endothelial function led by Prof HF Tse. The brachial artery was imaged using high-resolution ultrasound (7.5MHz linear array, Agilent Sonos 5500; Philips, Andover, MA). Arterial diameter and flow velocity were measured. Scans were taken at baseline, after tourniquet inflation for 5 minutes, and after sublingual glyceryl trinitrate spray. The percentage change in brachial artery diameter following reactive hyperaemia compared with the baseline was calculated. The coefficient of variation of FMD determination was 5%. Complete data were available for 245 subjects. Among these subjects, 151 had hypertension, 80 had diabetes mellitus and 240 were on lipid lowering therapy. Our results showed that plasma lipocalin-2 correlated with serum creatinine (ρ=0.23, p<0.001) but not FMD (ρ=0.005, p=0.94). Lipocalin-2 levels were lower in subjects on lipid lowering therapy (p=0.023). FMD correlated inversely with age (ρ=-0.14, p=0.03). Diabetes was associated with a lower FMD (p=0.044). Thus, surprisingly, the plasma level of lipocalin-2 was unrelated to FMD, an index of endothelial function. Endothelial function varies even within an individual, and is influenced by factors such as ageing and diabetes as found in our study, and also by diet and drugs. Most of the study subjects were on lipid lowering therapy, and those who had hypertension and diabetes were treated. Control of risk factors could have a favourable effect on endothelial function. The correlation of lipocalin-2 with creatinine is consistent with it being a marker of renal injury. This study was planned before the deamidation of lipocalin-2 was elucidated; it is possible that it correlates better with endothelial function. Deamidated lipocalin-2 and cardiovascular risk factors Accordingly, we went beyond the original objectives of the proposal to conduct a further study on deamidated lipocalin-2 in man. We previously demonstrated in man and mouse that lipocalin-2 is unstable in plasma and is modified by polyamination. Polyaminated lipocalin-2 is rapidly cleared whereas deamidated lipocalin-2 is not, so the latter may accumulate in arteries and induce endothelial dysfunction. We think that activation of lipocalin-2 by deamidation is a key mechanism mediating obesity-induced inflammation and endothelial dysfunction. Therefore we measured polyaminated and deamidated lipocalin-2 in normal healthy people in the community and determined their relationships with cardiovascular risk factors. One hundred normal healthy adults, comprising 59 men (mean age 47.7±7.5 years) and 41 women (mean age 48.2±7.2 years), were studied with informed consent. Blood pressure was measured three times using an automated sphygmomanometer, and a questionnaire was used to record the subjects’ medical history and lifestyle. The serum level of non-polyaminated lipocalin-2 was measured and was found to correlate positively with body mass index, waist circumference, heart rate, diastolic blood pressure and triglyceride levels in the blood, but negatively with circulating adiponectin. This provides the first human evidence that non-polyaminated lipocalin-2 may be involved in cardiovascular disease development. | |||||||||||||||
| Summary of objectives addressed: |
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| Research Outcome | ||||||||||||||||
| Major findings and research outcome: | Animal studies Amelioration of vascular inflammation, endothelial dysfunction and hypertension in lipocalin-2 knockout mice We found that lipocalin-2, in particular, its deamidated from, acts together with a high fat diet to cause accumulation of lipocalin-2 in the arterial wall, provoking inflammation and endothelial dysfunction. Mice deficient in lipocalin-2 are resistant to these harmful effects and the elevation of blood pressure caused by high fat diet. Linoleic acid treatment facilitated the deamidation of lipocalin-2, reduced its interactions with matrix metalloproteinase 9 (MMP9), and increased its circulating level and its accumulation in the arterial wall. We discovered that lipocalin-2 caused vascular inflammation through neutrophil activation. Lipocalin-2 knockout mice had reduced mineralocorticoid receptor expression and development of hypertension and fibrotic changes in the heart and kidneys. Human studies Lipocalin-2 and hypertension Over a median follow-up of 5.4 years, the baseline level of lipocalin-2 in plasma was found to be a significant independent predictor of new-onset hypertension. Lipocalin-2 and endothelial function We found to our surprise that the plasma level of lipocalin-2 was unrelated to FMD, an index of endothelial function. Instead, plasma lipocalin-2 correlated with serum creatinine, while lower FMD was associated with diabetes and advancing age. Deamidated lipocalin-2 and cardiovascular risk factors The serum level of non-polyaminated lipocalin-2 was measured in normal healthy adults and was found to correlate positively with obesity, triglyceride level and blood pressure, and inversely with adiponectin. This provides the first human evidence that non-polyaminated lipocalin-2 may be involved in cardiovascular disease development. | |||||||||||||||
| Potential for further development of the research and the proposed course of action: |
Having shown that deamidated lipocalin-2 plays a role in vascular inflammation, endothelial dysfunction and hypertension, further experiments are needed to understand the mechanisms whereby lipocalin-2 causes atherosclerosis and to demonstrate the therapeutic potential of suppressing lipocalin-2. We planned the study of the relationship between lipocalin-2 and endothelial function before we discovered the role of deamidated lipocalin-2. It is worth measuring the deamidated form, which we think is more closely related to the pathogenesis of atherosclerosis. | |||||||||||||||
| Layman's Summary of Completion Report: | Lipocalin-2 is a blood protein the function of which is to mop up excess iron and also to deprive bacteria of iron. Our studies in mice deficient in lipocalin-2 showed that there is less inflammation and dysfunction in arteries, and lower blood pressure. In man, we found that the blood level of lipocalin-2 correlated with obesity and blood pressure. In a cohort of normal people without hypertension to start with, a high baseline blood level of lipocalin-2 was a predictor of the development of hypertension 5 years later. To our surprise, we did not find a significant relationship between the blood level of lipocalin-2 and artery dysfunction in our cardiac patients, but that could have been due to the fact that they were taking medications to control their blood pressure and lipids. We discovered that the deamidated form of lipocalin-2 stays in the circulation longer than the native form and may be the culprit causing cardiovascular disease. Further studies will be conducted to understand the implications of this novel finding. | |||||||||||||||
| 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.): |
Parts of the animal studies were performed in collaboration with researchers from the Institut de Recherches Servier, Suresnes, France. The study of non-polyaminated lipocalin-2 in humans was carried out in collaboration with researchers from the University of Southern Denmark and the Odense University Hospital. | |||||||||||||||
| SCREEN ID: SCRRM00542 |