Sensitivity,specificity of biochemical markers for early prediction of endothelial dysfunction in atherosclerotic obese subjects

BackgroundThe obesity increased incidence of diabetes, hypertension and atherosclerosis and rate of morbidity and mortality. The main cause of atherosclerosis is endothelial dysfunction and formation of foam cells and macrophage that lead to unfavorable complications. This study evaluated specific biomarkers for endothelial dysfunction as sensitive indices for early predication of atherosclerosis in obese subjects.Study DesignOne hundred fifty male age and sex matching were included in the current study divided into three groups according to body mass index (BMI): Control (BMI ≤ 22), obese (BMI> 28) and obese with atherosclerosis (BMI> 28). Fasting serum was subjected for determination of adhesion molecules, sICAM-1, sVCAM-1, E-selectin, oxo-LDL and 8-iso-PGF2α by ELISA technique.ResultsData obtained showed that, a significant elevation of serum inflammatory markers CRP, IL-6 and TNF-α and adhesion molecules sICAM-1 (p<0.001) with sensitivity 96%, sVCAM-1 (p <0.01) with sensitivity 92%, E-selectin (p<0.001) with sensitivity 94%, oxo-LDL (p <0.05) and 8-iso-PGF2α (p < 0.001) with sensitivity 97% in obese with atherosclerosis compared with obese and control.ConclusionThe levels of serum adhesion molecules contributed in the pathogenesis of endothelial dysfunction can be used as sensitive biomarkers for early prediction of atherosclerosis in obese subjects.     

Establishment of a rat model with diet-induced coronary atherosclerosis

Coronary atherosclerotic disease is a serious disease in humans, but no suitable animal model is available currently for further studies. We used apolipoprotein E gene knockout (ApoE KO) rats to induce hypercholesterolemia through a special high cholesterol/bile salt diet (Paigen diet), then analyzed aortic and coronary atherosclerosis lesions and the myocardial injury in order to establish a novel small animal model of coronary atherosclerosis. Plasma cholesterol of ApoE KO rats increased 7.6-fold compared with wild-type rats after 8 weeks on the Paigen diet. After 10 to 12 weeks of subsisting on the Paigen diet, ApoE KO rats developed mild aortic atherosclerosis with severe coronary atherosclerosis. Hematoxilyn and eosin staining showed that 11 out of 12 ApoE KO male rats had right coronary artery atherosclerosis, 7 of them were>70% occluded. Oil Red O (Lipid Stain), Mac2 immuno-staining and Masson’s trichrome staining demonstrated substantial amounts of lipid, macrophages and collagen fibers in coronary atherosclerosis plaques. In addition, ApoE KO male rats had severe myocardial focal lesions with cholesterol ester as the main component in the lesions. In conclusion, ApoE KO rats developed severe hypercholesterolemia, coronary atherosclerosis and myocardial cholesterol ester deposition after subsisting on the Paigen diet and can be used as a novel animal model for studies on cholesterol metabolism and coronary atherosclerotic disease.     

血管内皮细胞功能检测方法的研究进展

血管内皮细胞(VEC)是多种心脑血管疾病或危险因子作用的靶器官,内皮功能损伤常伴发和加重心脑血管疾病,血管内皮功能障碍与动脉粥样硬化、高血压等一系列疾病和状态有关,是动脉粥样硬化最早期的改变,是冠心病患者未来心血管事件增加的一项独立的预测指标.如何早期评价血管内皮的功能对于临床防治血管性疾病具有重要意义.目前临床检测方法主要包括影像学检查、生物活性因子检测及循环内皮细胞和内皮祖细胞计数.     

兔高脂血症与血管内皮功能的关系

目的 观察一氧化氮(NO)和细胞间黏附分子-1(ICAM-1)在高脂血症及动脉粥样硬化(AS)动物模型中的表达,探讨高脂血症及AS与血管内皮功能和ICAM-1的关系.方法 采用高脂饮食饲养家兔建立高脂血症和AS动物模型,正常饮食组为对照组;两组动物于0、8和16周分别取静脉血,检测血清中胆固醇(TC)、低密度脂蛋白(LDL)、NO和ICAM-1水平,并观察TC和LDL与NO和ICAM-1的相关性;16周后处死动物,免疫组化法和RT-PCR检测ICAM-1在主动脉粥样硬化斑块中的表达.结果 饲养8周时,高脂饮食组血清TC和LDL明显增加(P＜0.01),NO和ICAM-1无明显变化;饲养16周时,NO水平明显下降(P＜0.01),ICAM-1水平明显增高(P＜0.01).TC和LDL与NO呈负相关,与ICAM-1呈正相关.结论 高脂血症及AS造成血管内皮功能失调,导致NO和ICAM-1的异常表达;NO和ICAM-1参与了AS的形成.     

血管树突状细胞在人主动脉粥样硬化早期病变中的分布

目的：探讨血管树突状细胞在人早期动脉粥样硬化(AS)病变中的分布模式。方法：人主动脉标本15例主要取自尸检和外科手术，常规连续切片，分别行HE及S100／CD1a免疫细胞化学染色，光镜下观察S100／CD1a阳性细胞分布情况。结果：15例HE染色标本中，2例正常，13例人动脉血管可见内膜的增厚及泡沫细胞等AS早期病理表现。9例S100／CD1a染色阳性，阳性率为69．2％。S100／CD1a阳性细胞分布在病变的内膜和外膜，外膜的S100／CD1a阳性细胞主要分布在滋养血管的周围。结论：在AS早期病变部位有血管树突状细胞的聚集，主要分布在病变血管的内膜和外膜，提示血管树突状细胞可能参与了AS早期的免疫反应。     

Imbalance in the production between vascular endothelial growth factor and endostatin in Kawasaki disease

To investigate whether an imbalance exists in the production between angiogenic and antiangiogenic growth factors in patients with Kawasaki disease (KD), we measured the serum levels of vascular endothelial growth factor (VEGF) and endostatin (ES) in 35 patients with KD, 15 patients with acute febrile diseases (disease controls) and 15 healthy children. KD patients had significantly higher VEGF levels and lower ES levels (P < 0.01) in the acute and subacute phases than the disease control and healthy children. KD patients with coronary artery lesions (CAL, n = 10) had significantly higher VEGF levels and lower ES levels (P < 0.05) in the subacute and convalescent phases than those without CAL (n = 25). The ratios of VEGF/ES in sera of KD patients with CAL were significantly higher (P < 0.05) in the acute and convalescent phases compared to those without CAL. Furthermore, the occurrence of CAL significantly correlated with the VEGF/ES ratio above 10 x 10(-3) in the subacute phase of KD (Odds ratio 17.25, P = 0.005). The findings in the present study indicate that an imbalance exists in the production between VEGF and ES in patients with KD while also suggesting that KD patients with a high VEGF/ES ratio have a significantly greater risk of CAL involvement.     

无血清单层细胞诱导法培养猪诱导多能性干细胞定向分化为血管内皮细胞

背景:诱导多能性干细胞是一种新型的干细胞来源,具有多向分化潜能。猪作为人类心血管及代谢性疾病研究的良好模型,对其多能性细胞向血管内皮细胞定向分化体系的建立,将为创建心血管疾病模型提供保障。目的:建立一种无血清单层诱导分化方法,使猪诱导多能性干细胞定向分化为CD31阳性血管内皮细胞,并对获得的内皮细胞进行鉴定。方法:使用CHIR99021、骨形态发生蛋白4、碱性成纤维生长因子、血管内皮生长因子等对猪诱导多能性干细胞和人胚胎干细胞进行单层细胞诱导分化,使干细胞经历3个分化阶段最终成为血管内皮细胞,检测并比较分化过程中2种干细胞的胚层标记基因的表达,对流式分选后获得的血管内皮细胞进行鉴定。结果与结论:①通过无血清单层细胞诱导法从猪诱导多能性干细胞定向诱导分化获得的内皮细胞具有与猪永生化主动脉血管内皮细胞类似的基因表达模式及生物学功能——能够吸收低密度脂蛋白并在Matrigel上形成微血管样结构;②该诱导体系下,猪诱导多能性干细胞的分化效率高于人胚胎干细胞的分化效率,这可能与诱导过程中2种细胞谱系基因表达模式上的差异相关。     

Characterization of tachykinin receptors in endothelial cells of porcine artery

The binding of iodine-labeled Bolton-Hunter substance P (¹²⁵I-BHSP) to porcine endothelial cell membranes was examined. The endothelial cells had a single high-affinity binding site with a dissociation constant of 0.10 nM, and a maximum number of binding sites of 52.2 fmol/mg protein. The relative potencies of various tachykinins to displace the binding of 47 pM ¹²⁵I-BHSP suggested that endothelial cells of porcine aorta contain the NK-1 subtype of tachykinin receptor. A GTP analogue, guanyl-5′-yl imidodiphosphate, induced marked reduction in the number of ¹²⁵I-BHSP binding sites suggesting that these binding sites are coupled with GTP-binding protein.     

循环血管内皮祖细胞血管修复作用的研究进展

循环内皮祖细胞在血管损伤的情况下,可归巢到受损区域,增殖分化为成熟内皮细胞,并分泌营养因子,促进内皮新生。但是,由于其干细胞特性,在微环境改变的情况下,可能无法分化为功能细胞,甚至表达纤维化、成骨分化等表型,加速血管损伤。因此,纯化单一细胞群,稳定微环境,明确病程是决定循环内皮祖细胞发挥积极作用的关键。     

简易高效分离培养大鼠胸主动脉血管内皮细胞方法的建立

目的 建立一种高效分离、培养大鼠胸主动脉血管内皮细胞的方法.方法 用Wistar大鼠(200 ～ 250 g),无菌条件下开胸取胸主动脉,去除血管外膜,制备长约1.0～1.5 mm动脉环.将血管环垂直置于干燥的培养皿中,并向血管环内加入含有10％胎牛血清的DMEM/F12培养液、静止培养;24h血管环贴壁后,加入培养液...     

不同剂量阿托伐他汀和停服后对肥胖高胆固醇血症患者 血管内皮功能和炎症因子的影响

目的：研究在肥胖高胆固醇血症患者中应用不同剂量阿托伐他汀及停服后对血管内皮功能和炎症因子的影响,探讨其可能的机制。方法：观察入选的55名肥胖高胆固醇血症患者和58名健康者肱动脉内皮依赖性舒张功能,并检测血清中NO,可溶性E 选择素（SE）及高敏C反应蛋白（hs CRP）含量的变化。将55名肥胖高胆固醇血症患者随机双盲分成两组：A组27例,在常规饮食控制基础上予阿托伐他汀10 mg/d;B组28例,在常规饮食控制基础上予阿托伐他汀20 mg/d。8周1疗程,观察两组治疗前、治疗后及停药1周后上述指标及血脂的变化。结果：阿托伐他汀治疗8周后可明显降低肥胖高胆固醇血症患者总胆固醇（TC）、低密度脂蛋白胆固醇（LDL C）、SE和hs CRP水平（均P<0.01）,明显提高NO水平（P<0.01）,改善肱动脉内皮依赖性舒张功能（P<0.05或P<0.01）;其中B组较A组上述改变更显著（P<0.05）。停药1周后两组TC和LDL C水平均较治疗8周时明显升高(P<0.05),但仍低于治疗前水平(P<0.05或P<0.01);两组NO和肱动脉内皮依赖性舒张功能均明显下降,恢复至治疗前水平(P>0.05),两组SE和hs CRP均明显升高,也恢复至治疗前水平(P>0.05)。结论：对肥胖高胆固醇血症患者应用阿托伐他汀20 mg/d较10 mg/d更能有效控制血脂水平,改善内皮功能和抑制炎症反应。而突然终止阿托伐他汀治疗可在1周内完全逆转该药对血管内皮功能的改善作用和炎症反应的抑制作用,停药对血管内皮功能和炎症因子的影响是非胆固醇依赖性作用。     

Reduction in visceral adiposity is highly related to improvement in vascular endothelial dysfunction among obese women: an assessment of endothelial function by radial artery pulse wave analysis

Because obesity is frequently complicated by other cardiovascular risk factors, the impact of a reduction in visceral adiposity on vascular endothelial dysfunction (VED) in obese patients is difficult to determine. In the present study, we evaluated the impact of a reduction in visceral adiposity on VED in obese women. Thirty-six premenopausal obese women (BMI >/= 25 kg/m2) without complications were enrolled in the study. VED was evaluated by determining the augmentation index (AIx) from radial artery pulse waves obtained by applanation tonometry. Changes in AIx in response to nitroglycerin- induced endothelium-independent vasodilatation (DeltaAIx-NTG) and in response to salbutamol administration (DeltaAIx-Salb) were determined before and after weight reduction. After a 12-week weight reduction program, the average weight loss was 7.96 +/- 3.47 kg, with losses of 21.88 +/- 20.39 cm2 in visceral fat areas (p < 0.001). Pulse wave analysis combined with provocative pharmacological testing demonstrated preserved endothelium-independent vasodilation in healthy premenopausal obese women (DeltaAIx-NTG: 31.36 +/- 9.80% before weight reduction vs. 28.25 +/- 11.21% after weight reduction, p > 0.1) and an improvement in endothelial-dependent vasodilation following weight reduction (DeltaAIx-Salb: 10.03 +/- 6.49% before weight reduction vs. 19.33 +/- 9.28% after reduction, p < 0.001). A reduction in visceral adipose tissue was found to be most significantly related to an increase in DeltaAIx-Salb (beta=-0.57, p < 0.001). A reduction in visceral adiposity was significantly related to an improvement in VED. This finding suggests that reduction of visceral adiposity may be as important as the control of other major risk factors in the prevention of atherosclerosis in obese women.     

Biocompatibility of porcine small intestinal submucosa and rat endothelial progenitor cells in vitro

Objective: This study investigated the biocompatibility of the small intestinal submucosa (SIS) and endothelial progenitor cells (EPCs) by co-cultivating EPCs and SIS in vitro and observing EPC growth on the SIS. Methods: The porcine SIS was prepared and bone marrow mononuclear cells (BMMNCs) were isolated from 3 or 4-week old male SD rats. Cellular morphology was observed by light microscopy and scanning electron microscopy (SEM) and viabilities by the MTT assays. Endothelial progenitor cells (EPCs) were phenotyped by immunocytochemistry, immunofluorescence microscopy and flow cytometry. Vascular lumen formation was evaluated by the Matrigel tube formation assays. EPCs were seeded onto the SIS and production of angiogenin-1 and endothelial cell growth factor (VEGF) by EPCs was examined by ELISA and immunoblotting assays. Results: Light microscopy and SEM showed that the mechanically and chemically treated small intestinal submucosa was composed of cell-free extracellular matrix. Immunohistochemistry, and flow cytometry revealed that the EPCs expressed appropriate surface markers including CD34, CD133, and VEGFR-2. Furthermore, the EPCs formed lumen-like structures and the SIS significantly enhanced the growth of EPCs in vitro. Conclusion: SIS has good biocompatibility with EPCs. SIS pre-seeded with EPCs can be potentially applied as an alternative scaffold material in artificial blood vessel prosthesis.     

Nicotine potentiates vascular endothelial growth factor expression in balloon-injured rabbit aortas

Both nicotine and vascular endothelial growth factor (VEGF) have been proposed to play an important role in the development and progression of atherosclerosis. In vitro and ex vivo studies have demonstrated that nicotine significantly stimulates VEGF expression in several cell types. This study examined the effects and the mechanisms of nicotine on the expression of VEGF in a rabbit model of balloon-injured aortas. Forty-eight male New Zealand white rabbits were randomly divided into sham, control, nicotine, and nicotine plus hexamethonium (nicotine–hex) groups. Balloon catheter denuding injury iliac artery was performed in control, nicotine, and nicotine–hex animals fed with a high-cholesterol diet beginning 2 weeks before operation. Twenty-four hours after surgery, nicotine (0.05 μg/kg) or nicotine (0.05 μg/kg) and hexamethonium (6 mg/kg) was administered daily by intramuscular injection for 3 weeks in nicotine and nicotine–hex groups, respectively. Sham and control rabbits received an identical volume of phosphate-buffered saline injection, but without nicotine or hexamethonium. VEGF protein expression and intimal cell proliferation in balloon-injured aortas were determined by enzyme-link immunosorbent assay, immunohistochemistry, and Western blot analysis. Six rabbits died during the experiment. The remaining 42 rabbits were included in the study. VEGF protein expression in nicotine group was significantly higher than that in control group (P < 0.01). VEGF positive staining was seen in vascular endothelial cells, vascular smooth muscle cells, and infiltrative inflammatory cells. The number of the proliferative cells in intima was also significantly higher in nicotine group than in control group (P < 0.01). Hexamethonium, a nonselective antagonist of nicotinic acetylcholine receptors (nAChRs), significantly inhibited nicotine-induced VEGF protein expression (P < 0.01). The present study shows that intramuscular administration of nicotine markedly potentiates the expression of VEGF protein in balloon-injured rabbit aortas, which appears to be mediated through nAChRs.     

The effect of acute exercise on endothelial function following a high-fat meal

The transient impairment of endothelial function following a high-fat meal is well established. Brachial artery flow-mediated dilation (FMD) decreases between 2 and 6 h post ingestion. Whether this impairment can be reduced with acute aerobic exercise has not been investigated. The purpose of this study was to investigate if a single sustained aerobic exercise session can counteract the postprandial attenuation in brachial artery FMD associated with the ingestion of a high-fat meal. Eight apparently healthy adults (five men, three women), age 25.5 ± 0.8 years, performed three treatment conditions in a counter-balanced design: (1) low-fat meal alone (LFM), (2) high-fat meal alone (HFM), and (3) one session of aerobic exercise presented 2 h after ingesting a high-fat meal (HFM-EX). The examination of brachial artery FMD was performed at baseline and 4 h following the ingestion of the meal for each treatment condition. A 3 × 2 (treatment × time) repeated measures ANOVA exhibited a significant interaction (P = 0.019). Preprandial FMDs were similar (P = 0.863) among all three treatment conditions. The FMDs following the LFM (7.18 ± 1.31%) and HFM-EX (8.72 ± 0.94%) were significantly higher (P = 0.001) than the FMD following the HFM (4.29 ± 1.64%). FMD was significantly elevated above preprandial values following the HFM-EX (5.61 ± 1.54 to 8.72 ± 0.94%, P = 0.005) but was unchanged following the LFM (6.17 ± 0.94 to 7.18 ± 1.31%, P = 0.317) and the HFM (5.73 ± 1.23 to 4.29 ± 1.64%, P = 0.160). These findings suggest that a single aerobic exercise session cannot only counteract the postprandial endothelial dysfunction induced by the ingestion of a high-fat meal, but also increase brachial artery FMD in apparently healthy adults.     

Reduction of angiotensin A and alamandine vasoactivity in the rabbit model of atherogenesis: differential effects of alamandine and Ang(1‐7)

Novel treatments are necessary to reduce the burden of cardiovascular disease (CVD). Alamandine binds to MrgD and is reported to induce vasodilation via stimulation of endothelial nitric oxide synthase (eNOS), but its role in atherogenic blood vessels is yet to be determined. To determine the vasoactive role of alamandine and its precursor AngA in diseased aorta, New Zealand White rabbits were fed a diet containing 1% methionine + 0.5% cholesterol + 5% peanut oil for 4 weeks (MC, n = 5) or control (n = 6). In abdominal aorta, alamandine (1 μM) was added 30 min before a dose–response curve to angiotensin II or AngA (1 nM–1 μM), and immunohistochemistry was used to identify MrgD receptors and eNOS. The thoracic aorta, renal, carotid and iliac arteries were mounted in organ baths. Rings were precontracted with phenylephrine, then a bolus dose of alamandine (1 μM) was added 10 min before a dose–response curve to acetylcholine (0.01 μM–10 μM). The MrgD receptor was localized to normal and diseased aorta and colocalized with eNOS. In control but not diseased blood vessels, alamandine enhanced acetylcholine‐mediated vasodilation in the thoracic aorta and the iliac artery (P < 0.05) and reduced it in the renal artery (P < 0.05). In control abdominal aorta, AngA evoked less desensitization than AngII (P < 0.05) and alamandine reduced AngA‐mediated vasoconstriction (P < 0.05). In MC, AngA constriction was markedly reduced vs. control (P < 0.05). The vasoactivity of alamandine and AngA are reduced in atherogenesis. Its role in the prevention of CVD remains to be validated.     

The importance of a substantial elastic lamina subjacent to the endothelium in limiting the progression of atherosclerotic changes

This study examines the hypothesis that progressive intimal thickening and atherosclerosis in the larger pulsatile arteries arise from failure to maintain, subjacent to the endothelial cells, a substantial elastin membrane, a component which has been shown to be of special structural significance. The internal thoracic arteries of 293 subjects of all ages up to 60 years were compared histologically with the anterior descending coronary arteries of the same individuals by light- and electronmicroscopy and immunoperoxidase staining for macromolecules. The internal thoracic arteries usually developed a new robust reduplicated internal elastic lamina at an early age, no further intimal thickening, and no significant entry of lipid or cells to the intima. The coronary arteries showed areas of rapid intimal thickening with poor and incomplete reduplicated internal elastic laminae, entry of lipid, macrophages, and other cells to the intima. The reduplicated internal elastic laminae appeared to be formed primarily by the endothelial cells themselves. An elastin membrane subjacent to the endothelial cells appears to be essential. It provides a secure attachment for the cells and a barrier to the entry of macromolecules and cells to the intima. Its absence is associated with progressive intimal thickening and atherosclerosis.