Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1^+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1^+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE^-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.     

用siRNA沉默人血红素加氧酶-1基因降低胆红素水平

拟通过RNA干扰技术特异下调人血红素加氧酶-1(human heme oxygenase-1,hHO-1)基因的表达,减少hHO-1的产量从而降低胆红素的产生,探讨在胆红素产生前就阻断其产生,为临床早期防治新生儿高胆红素血症及胆红素中毒性脑病探索一种新的有效手段。针对hHO-1基因设计并化学合成三对小分子干扰RNA(small interfering RNA,siRNA)。采用脂质体转染法将siRNA转染入人肝脏细胞株HL-7702;荧光显微镜检测siRNA转染细胞的效率;转染siRNA1~2天后经RT-PCR和Western印迹方法检测hHO-1表达水平和蛋白质量;并采用HO-1诱导剂血红素诱导或hHO-1表达质粒转染细胞以上调hHO-1表达,检测siRNA干扰后hHO-1产量和酶活性。结果显示:设计的三对siRNA能不同程度的特异下调hHO-1表达,筛选获得抑制效果最佳的siRNA-3。siRNA-3抑制hHO-1呈现浓度与时间依赖性。与非特异对照siRNA及未处理组比较,血红素诱导和hHO-1表达质粒转染均能上调HL-7702细胞内hHO-1表达,提高hHO-1产量,但转染siRNA-3后hHO-1表达明显抑制,同时hHO-1活性随着基因表达下调而下降。实验表明设计合成的siRNA-3抑制效果明显。siRNA-3通过降解hHO-1,减少hHO-1产量,降低酶活性,最终减少胆红素产生,从而使RNA干扰技术成为降低新生儿高胆红素血症和胆红素中毒性脑病发生的一种候选方法。     

Sp1 Regulates Chromatin Looping between an Intronic Enhancer and Distal Promoter of the Human Heme Oxygenase-1 Gene in Renal Cells

HO-1 (heme oxygenase-1) is an inducible microsomal enzyme that catalyzes the degradation of pro-oxidant heme. The goal of this study was to characterize a minimal enhancer region within the human HO-1 gene and delineate its role in modulating HO-1 expression by participation with its promoter elements in renal epithelial cells. Deletion analysis and site-directed mutagenesis identified a 220-bp minimal enhancer in intron 1 of the HO-1 gene, which regulates hemin-mediated HO-1 gene expression. Small interfering RNA, decoy oligonucleotides, site-directed mutagenesis, and chromatin immunoprecipitation assays confirmed the functional interaction of Sp1 with a consensus binding sequence within the 220-bp region. Mutations of regulatory elements within the −4.5 kb promoter region (a cyclic AMP response and a downstream NF-E2/AP-1 element, both located at −4.0 kb, and/or an E-box sequence located at −44 bp) resulted in the loss of enhancer activity. A chromosome conformation capture assay performed in human renal epithelial (HK-2) cells demonstrated hemin-inducible chromatin looping between the intronic enhancer and the −4.0 kb promoter region in a time-dependent manner. Restriction digestion with ApaLI (which cleaves the 220-bp enhancer) led to a loss of stimulus-dependent chromatin looping. Sp1 small interfering RNA and mithramycin A, a Sp1 binding site inhibitor, resulted in loss of the loop formation between the intronic enhancer and the distal HO-1 promoter by the chromosome conformation capture assay. These results provide novel insight into the complex molecular interactions that underlie human HO-1 regulation in renal epithelial cells.     

Risk Stratification of Patients with Peripheral Arterial Disease and Abdominal Aortic Aneurysm Using Aortic Augmentation Index

BackgroundCentral augmentation index (cAIx) is an indicator for vascular stiffness. Obstructive and aneurysmatic vascular disease can affect pulse wave propagation and reflection, causing changes in central aortic pressures.AimTo assess and compare cAIx in patients with peripheral arterial disease (PAD) and / or abdominal aortic aneurysm (AAA).MethodscAIx was assessed by radial applanation tonometry (Sphygmocor) in a total of 184 patients at a tertiary referral centre. Patients were grouped as having PAD only, AAA only, or both AAA and PAD. Differences in cAIx measurements between the three patient groups were tested by non-parametric tests and stepwise multivariate linear regression analysis to investigate associations with obstructive or aneurysmatic patterns of vascular disease.ResultsIn the study sample of 184 patients, 130 had PAD only, 20 had AAA only, and 34 patients had both AAA and PAD. Mean cAIx (%) was 30.5 ± 8.2 across all patients. It was significantly higher in females (35.2 ± 6.1, n = 55) than males (28.4 ± 8.2, n = 129), and significantly higher in patients over 80 years of age (34.4 ± 6.9, n = 22) than in those under 80 years (30.0 ± 8.2, n = 162). Intergroup comparison revealed a significant difference in cAIx between the three patient groups (AAA: 27.3 ± 9.5; PAD: 31.4 ± 7.8; AAA & PAD: 28.8 ± 8.5). cAIx was significantly lower in patients with AAA, higher in patients with both AAA and PAD, and highest in patients with PAD only (beta = 0.21, p = 0.006).ConclusionNon-invasive assessment of arterial stiffness in high-risk patients indicates that cAIx differs according to the pattern of vascular disease. Measurements revealed significantly higher cAIx values for patients with obstructive peripheral arterial disease than for patients with aneurysmatic disease.     

Reported High Salt Intake Is Associated with Increased Prevalence of Abdominal Aortic Aneurysm and Larger Aortic Diameter in Older Men

Background

Salt intake has been implicated in the pathogenesis of abdominal aortic aneurysm (AAA) through studies in rodent models but not previously studied in humans. The aim of this study was to examine the association between reported addition of salt to food and the prevalence of AAA.

Methods

A risk factor questionnaire which contained a question about salt intake was included as part of a population screening study for AAA in 11742 older men. AAA presence was assessed by abdominal ultrasound imaging using a reproducible protocol.

Results

The prevalence of AAA was 6.9, 8.5 and 8.6% in men who reported adding salt to food never, sometimes and always, respectively, p = 0.005. Addition of salt to food sometimes (odds ratio [OR]: 1.22, 95% confidence interval [CI]: 1.03–1.44) or always (OR: 1.23, 95% CI 1.04–1.47) was independently associated with AAA after adjustment for other risk factors including age, waist-hip ratio, blood pressure, history of hypertension, high cholesterol, angina, diabetes, myocardial infarction and stroke. Salt intake was also independently associated with aortic diameter (beta 0.023, p = 0.012). In men with no prior history of hypertension, high cholesterol, angina, myocardial infarction or stroke (n = 4185), the association between addition of salt to food sometimes (OR: 1.41, 95% CI 0.96–2.08) or always (OR: 1.52, 95% CI 1.04–2.22) and AAA remained evident.

Conclusion

Reported salt intake is associated with AAA in older men. Additional studies are needed to determine whether reducing salt intake would protect against AAA.     

银屑病的研究进展及其与血红素氧合酶1的关系

银屑病是一种免疫调节紊乱的慢性炎性皮肤疾病,多为T细胞功能异常所致,具有发病率高、易复发、病情顽固等特征。该病还与遗传及感染、饮食、压力等环境因素相关。目前,银屑病的治疗方法多种,但仍无法根治。研究显示,血红素氧合酶1(heme oxygenase 1,HO-1)在斑块型银屑病患者皮损处明显升高。HO-1具有抗炎、抗凋亡和改变增殖的作用,它可能通过调节氧化应激而介导银屑病的发生、发展进程。本文综述近年来的研究结果,讨论银屑病的发病机理及HO-1在该病治疗中的可能作用,旨在为临床治疗银屑病提供一些新的理论基础。     

Loss of Timp3 Gene Leads to Abdominal Aortic Aneurysm Formation in Response to Angiotensin II

Aortic aneurysm is dilation of the aorta primarily due to degradation of the aortic wall extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs), the proteases that degrade the ECM. Timp3 is the only ECM-bound Timp, and its levels are altered in the aorta from patients with abdominal aortic aneurysm (AAA). We investigated the causal role of Timp3 in AAA formation. Infusion of angiotensin II (Ang II) using micro-osmotic (Alzet) pumps in Timp3^−/− male mice, but not in wild type control mice, led to adverse remodeling of the abdominal aorta, reduced collagen and elastin proteins but not mRNA, and elevated proteolytic activities, suggesting excess protein degradation within 2 weeks that led to formation of AAA by 4 weeks. Intriguingly, despite early up-regulation of MMP2 in Timp3^−/−Ang II aortas, additional deletion of Mmp2 in these mice (Timp3^−/−/Mmp2^−/−) resulted in exacerbated AAA, compromised survival due to aortic rupture, and inflammation in the abdominal aorta. Reconstitution of WT bone marrow in Timp3^−/−/Mmp2^−/− mice reduced inflammation and prevented AAA in these animals following Ang II infusion. Treatment with a broad spectrum MMP inhibitor (PD166793) prevented the Ang II-induced AAA in Timp3^−/− and Timp3^−/−/Mmp2^−/− mice. Our study demonstrates that the regulatory function of TIMP3 is critical in preventing adverse vascular remodeling and AAA. Hence, replenishing TIMP3, a physiological inhibitor of a number of metalloproteinases, could serve as a therapeutic approach in limiting AAA development or expansion.     

腔内修复术治疗腹主动脉瘤的临床疗效分析

目的:评价腔内修复术(EVAR)治疗腹主动脉瘤(AAA)的临床疗效及安全性。方法:选择2008年12月至2013年12月收治的29例AAA患者,给予EVAR治疗,观察其围术期的疗效及血管破裂死亡、伤口愈合情况、截瘫、腔内隔绝术后并发症的发生情况和随访期疗效及血管破裂死亡、截瘫及内漏的发生情况。结果:29例手术均成功,1例术后3天出现右髂动脉支架内血栓、消化道出血及肝肾功能衰竭,行持续性血液净化好转出院。2例术区切口愈合延迟,9例术后发热,无在院死亡及截瘫病患。随访期间,1例术后30天死亡,死于肝肾功能衰竭;1例3个月出现肾功能不全;1例双下肢乏力,无截瘫发生。存活的28例患者复查增强CT见支架位置、形态良好,无移位及内漏发生。结论:EVAR具有成功率高、创伤小、恢复快等特点,且并发症少,治疗AAA安全有效。     

Different Long-Term Outcomes of Abdominal Aortic Aneurysm and Intracranial Aneurysm Models: Hemodynamic Change may also Play an Essential Role in the Initiation and Progression of Abdominal Aortic Aneurysm in Rabbits

Self-healing phenomenon was found in the periarterial elastase-induced abdominal aortic aneurysm (AAA) in rabbit. This kind of aneurysm model does not progress and heals spontaneously in the long term, which is quite different from the performance of AAA disease in human. In order to better mimic human AAA and overcome this shortcoming of traditional AAA model in rabbit, we studied the pathogenesis of cerebral aneurysm (CA) model in small animal, which shows an excellent long-term patency and progressive enlargement. We found that hemodynamic conditions, such as turbulence flow, high blood flow, and shear stress, play an important role in the formation and progression of CA. So, we hypothesize that hemodynamic change may also play an essential role in the initiation and progression of rabbit AAA, and self-healing will be overcome if hemodynamic condition changes by coarctation of infra-renal aorta after elastase incubation.     

Extracellular-Purine Metabolism in Blood Vessels (Part I). Extracellular-Purine Level in Blood of Patients with Abdominal Aortic Aneurysm

Adenosine and adenosine derivatives are the main regulators of purinoceptors (P1 and P2) mediated hemostasis and blood pressure. Since impaired hemostasis and high blood pressure lead to atherosclerosis and to the development of aneurysm, in this study we tested and compared the concentration of extracellular purines (e-purines) in the blood in of patients having abdominal aortic aneurysm with that from healthy volunteers. Whereas adenine nucleosides and nucleotides level in human blood plasma was analysed using reverse phase high performance liquid chromatography (HPLC), cholesterol concentration was estimated by an enzymatic assay. We did not find any correlation between e-purines concentration and the age of healthy volunteers. Furthermore, the sum level of e-purines (ATP, ADP, AMP, adenosine, and inosine) in the control group did not exceed 70 μM, while it was nearly two-fold higher in the blood of patients having abdominal aortic aneurysm, (123 μM). In a special case of people with Leriche Syndrome, a disease characterized by deep atherosclerotic changes, the e-purines level had further increased. Additionally, we also report typical atherosclerotic changes in the aorta using histological assays as well as total cholesterol rise. The significant rise in cholesterol concentration in the blood of the patients with abdominal aortas aneurysm, compared with the control groups, was not unique since 23% of the healthy people also exceeded the normal level of cholesterol. Therefore, our results strongly indicate that the estimation of e-purines concentration in the blood may serve as another indicator of atherosclerosis and warrant further consideration as a futuristic diagnostic tool.     

Toll-Like Receptor 4 Is Essential in the Development of Abdominal Aortic Aneurysm

Toll-like receptor (TLR) family plays a key role in innate immunity and various inflammatory responses. TLR4, one of the well-characterized pattern-recognition receptors, can be activated by endogenous damage-associated molecular pattern molecules such as high mobility group box 1 (HMGB1) to sustain sterile inflammation. Evidence suggested that blockade of TLR4 signaling may confer protection against abdominal aortic aneurysm (AAA). Herein we aimed to obtain further insight into the mechanism by which TLR4 might promote aneurysm formation. Characterization of the CaCl₂-induced AAA model in mice revealed that upregulation of TLR4 expression, localized predominantly to vascular smooth muscle cells (VSMCs), was followed by a late decline during a 28-day period of AAA development. In vitro, TLR4 expression was increased in VSMCs treated with HMGB1. Knockdown of TLR4 by siRNA attenuated HMGB1-enhanced production of proinflammatory cytokines, specifically interleukin-6 and monocyte chemoattractant protein-1 (MCP-1), and matrix-degrading matrix metalloproteinase (MMP)-2 from VSMCs. In vivo, two different strains of TLR4-deficient (C57BL/10ScNJ and C3H/HeJ) mice were resistant to CaCl₂-induced AAA formation compared to their respective controls (C57BL/10ScSnJ and C3H/HeN). Knockout of TLR4 reduced interleukin-6 and MCP-1 levels and HMGB1 expression, attenuated macrophage accumulation, and eventually suppressed MMP production, elastin destruction and VSMC loss. Finally, human AAA exhibited higher TLR4 expression that was localized to VSMCs. These data suggest that TLR4 signaling contributes to AAA formation by promoting a proinflammatory status of VSMCs and by inducing proteinase release from VSMCs during aneurysm initiation and development.     

Transition between Acute and Chronic Hepatotoxicity in Mice Is Associated with Impaired Energy Metabolism and Induction of Mitochondrial Heme Oxygenase-1

The formation of protein inclusions is frequently associated with chronic metabolic diseases. In mice, short-term intoxication with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to hepatocellular damage indicated by elevated serum liver enzyme activities, whereas only minor morphological changes are observed. Conversely, chronic administration of DDC for several weeks results in severe morphological damage, characterized by hepatocellular ballooning, disruption of the intermediate filament cytoskeleton, and formation of Mallory-Denk bodies consisting predominantly of misfolded keratins, Sqstm1/p62, and heat shock proteins. To evaluate the mechanistic underpinnings for this dichotomy we dissected the time-course of DDC intoxication for up to 10 weeks. We determined body weight change, serum liver enzyme activities, morphologic alterations, induction of antioxidant response (heme oxygenase-1, HO-1), oxidative damage and ATP content in livers as well as respiration, oxidative damage and the presence and activity of HO-1 in endoplasmic reticulum and mitochondria (mtHO-1). Elevated serum liver enzyme activity and oxidative liver damage were already present at early intoxication stages without further subsequent increase. After 2 weeks of intoxication, mice had transiently lost 9% of their body weight, liver ATP-content was reduced to 58% of controls, succinate-driven respiration was uncoupled from ATP-production and antioxidant response was associated with the appearance of catalytically active mtHO-1. Oxidative damage was associated with both acute and chronic DDC toxicity whereas the onset of chronic intoxication was specifically associated with mitochondrial dysfunction which was maximal after 2 weeks of intoxication. At this transition stage, adaptive responses involving mtHO-1 were induced, indirectly leading to improved respiration and preventing further drop of ATP levels. Our observations clearly demonstrate principally different mechanisms for acute and chronic toxic damage.     

Circulating S100A12 Levels Are Associated with Progression of Abdominal Aortic Calcification in Hemodialysis Patients

Vascular calcification is an important factor associated with mortality in dialysis patients. Recently, soluble receptor for advanced glycation end product (sRAGE) and extracellular RAGE binding protein S100A12 (EN-RAGE) have been reported to be involved in the process of vascular calcification. Therefore, we investigated whether sRAGE and S100A12 are useful indicators of progression of abdominal aortic calcification in hemodialysis (HD) patients. We analyzed annual changes in vascular calcification score (VCS) for up to 4 years, compared to clinical and biological parameters in 149 HD patients. VCS was assessed annually using plain X-ray images of the lateral lumbar spine. The progression group was defined as patients with an increase in VCS more than 1 point each year on average during the observation period. Time-averaged concentrations were also evaluated to examine the association between biological parameters and changes in VCS. The patients had a mean age of 58.59 ± 12.93 years; 53.7% were male, and 45% were diabetic. The VCS increased in 55 patients; the mean increase was 1.60 ± 2.91 points. In a stepwise multivariate logistic analysis, we found that higher levels of S100A12 were significantly associated with progression of VCS (odds ratio [OR], 2.622; 95% confidence interval [CI], 1.371–5.016; P = 0.004). The relationship between sRAGE and VCS was not statistically significant (OR, 0.644; 95% CI, 0.302–1.374; P = 0.255). Our findings suggest that serum levels of S100A12 are associated with progression of abdominal aortic calcification in HD patients, independent of sRAGE level.     

Plasma Heme Oxygenase-1 Concentration Is Elevated in Individuals with Type 2 Diabetes Mellitus

Background

Circulating concentrations of heme oxygenase-1 (HO-1) have been recently reported to be elevated in several chronic disorders. However, no study has ever examined the association between circulating HO-1 concentrations and type 2 diabetes mellitus (T2DM).

Methods and Findings

581 cases with newly-diagnosed T2DM (New-T2DM) and 611 comparison controls were recruited in this two-phase case-control study, comprising 420 cases and 429 controls collected in the first phase study and 161 cases and 182 controls in the second phase replication study. Analyses, using both separated data and combined data from the two-phase studies, show that plasma HO-1 concentrations were significantly increased in New-T2DM cases compared to controls (P<0.001). Plasma HO-1 concentrations were significantly correlated with plasma glucose concentrations, HOMA-beta and HOMA-IR (P<0.001). After adjustment for age, sex, BMI and family history of diabetes, the ORs for New-T2DM in the highest quartile of plasma HO-1 concentrations, compared with the lowest, was 8.23 (95% CI 5.55–12.21; P for trend <0.001). The trend remained significant after additional adjustment for fasting plasma glucose/insulin, HOMA-beta/HOMA-IR, TC/TG, smoking, drinking and history of hypertension, and even in further stratification analysis by age, sex, BMI, smoking, drinking and history of hypertension.

Conclusions

Elevated plasma HO-1 concentrations are associated with higher ORs for New-T2DM, which add more knowledge regarding the important role of oxidative stress in T2DM. More consequent studies were warranted to confirm the clinical utility of plasma HO-1, especially in diagnosis and prognosis of T2DM and its complications.