糖尿病大鼠冠状小动脉超氧阴离子产生量的观察

目的探讨糖尿病大鼠高糖状态下冠状小动脉超氧阴离子(O2-)产生量,阐明高糖状态对O2-产生的影响。方法用链脲佐菌素制备大鼠糖尿病模型,通过手术显微镜及显微手术器械分离大鼠冠状动脉,冷冻切片制备部分标本,用免疫荧光技术测定糖尿病及正常大鼠冠状动脉O2-产生量。结果糖尿病模型组大鼠冠状动脉O2-产生量与正常对照组大鼠及模型组预先加入自由基清除剂组相比明显增加;冷冻切片冠状动脉横切面荧光染色结果表明糖尿病大鼠高糖状态下血管平滑肌与内皮O2-产生均增多。结论高糖能刺激冠状小动脉产生过量的O2-,参与糖尿病状态下的氧化应激,可能与糖尿病微血管并发症密切联系。     

碘缺乏和碘过量对大鼠心肌肌球蛋白重链基因表达的影响

目的观察碘缺乏和碘过量对大鼠心肌肌球蛋白重链(MHC)基因表达的影响。方法Wistar大鼠随机分为低碘(LI)组、适碘(NI)组、5、10、50、100倍高碘(5HI、10HI、50HI、100HI)组,饲养6个月。采用化学发光方法测定血中甲状腺激素(TH)水平,记录大鼠心电图,心脏指数,RT-PCR法检测心肌肌球蛋白重链α和β(α-MHC、β-MHC)mRNA的表达。结果与NI组相比,LI组血清TH水平降低(P<0．01),心脏指数增大(t=2．973,P<0．01),心肌细胞α-MHC mRNA表达量降低(t=4．382,P<0．01),β-MHC mRNA表达量增高(t=5．843,P<0．01)。各HI组血清TH水平较NI组有下降趋势,10H1、50HI、100HI组血清TT3与NI组相比差异有统计学意义(t=4．901、4．838、2．406,P<0．01或0．05),10HI、50HI组血清TT4与NI组相比差异有统计学意义(t=2．162、2．348,P<0．05);但心电图各波段和心脏指数未见明显改变:α-MHC mRNA表达量随摄入含碘量的升高而呈下凋趋势,但与NI组相比差异无统计学意义(P>0．05),而50HI和100HI组β-MHC mRNA表达量显著上调(t=2．632、4．127．P<0．05或0．01)。结论碘缺乏和碘过量均可导致Wistar大鼠甲状腺功能减退(甲减),影响心脏受TH调节的靶基因的表达量,进而影响心脏的功能状态。     

磷酯酰肌醇-3-激酶、丝氨酸／苏氨酸蛋白激酶在2型糖尿病大鼠骨组织中的表达及意义

目的探讨磷酯酰肌醇-3-激酶（P13K）、丝氨酸／苏氨酸蛋白激酶（Akt）在糖尿病大鼠骨组织中的表达及意义。方法将50只体重180—200g的6月龄雌性Wistar大鼠按随机数字表法分为对照组（n=24）和2型糖尿病组（n=26）。2型糖尿病组大鼠予高糖高脂饮食喂养8周后,腹腔注射链脲佐菌素30mg／kg,对照组大鼠正常饮食。糖尿病成模后12周两组大鼠分别行腰椎骨密度测定;应用逆转录聚合酶链反应（RT—PCR）法检测骨组织P13K、Aktl和Akt2mRNA的相对表达。组间比较采用t检验。结果对照组大鼠骨密度高于糖尿病组,差异有统计学意义[（0．079±0．034）比（0．060±0．017）g／cm2,t=2．499,P〈0．05];同时,对照组大鼠骨组织P13K、Aktl、Akt2mRNA均高于糖尿病组（分别为：0．65±0．05比0．51±0．04、0．756±O．031比0．694±0．017、0．77±0．04比0．66±0．04）,差异均有统计学意义（t=4．969、3．924、4．515,均P〈0．01）。结论2型糖尿病大鼠骨组织P13K、Aktl及Akt2表达低于正常,这可能是2型糖尿病骨质疏松发生的分子生物学机制之一。     

大鼠颈总动脉零应力状态与内皮型一氧化氮合酶的关系

目的探讨不同月龄大鼠颈总动脉零应力状态变化与内皮型一氧化氮合酶（eNOS）蛋白表达的关系。方法取30只3、12和24个月龄SD大鼠（每组10只）。处死大鼠后，迅速取下左右两侧颈总动脉。左侧颈总动脉置人4℃氧合Kreb’s液（pH=7．4）中，用于各组大鼠颈总动脉血管的零应力状态张开角的测量。右侧颈总动脉置人4％多聚甲醛溶液，采用组织形态学方法结合计算机图像分析，对颈总动脉组织形态学及定量形态学进行观察，并应用免疫组织化学染色技术，观察各组大鼠血管壁eNOS蛋白表达情况。结果随着大鼠月龄增高，成年大鼠颈总动脉发生明显重构，血管内膜增生明显，并伴有eNOS蛋白表达的降低。①3个月龄大鼠颈总动脉内皮细胞和中膜平滑肌细胞eNOS阳性表达数分别为10．1±2．5和79±8，显著高于12（6．7±1．4,64±8）和24个月龄大鼠（1．9±0．9，30±5），P〈0．01；②3、12和24个月龄大鼠颈总动脉张开角分别为（80±6）度、（110±6）度和（121±4）度，12和24个月龄大鼠颈总动脉的张开角都显著大于3个月龄大鼠的张开角（P〈0．01）；③应用相关分析表明，不同月龄大鼠血管内皮细胞和中膜平滑肌细胞eNOS阳性表达数量与血管张开角变化呈负相关（r=-0．8089，r=-0．7560，P〈0．05）。结论无论在血管形态学还是在零应力状态方面，不同月龄大鼠颈总动脉都存在着显著的差异，而且血管壁组织eNOS蛋白表达与大鼠颈总动脉零应力状态的改变呈显著负相关。     

2型糖尿病大鼠急性心肌缺血对心肌新生血管的影响

目的探讨糖尿病急性心肌缺血对心肌新生血管生成的影响及其机制。方法清洁级健康雄性8周龄sD大鼠52只,任意选取其中20只以高脂喂养联合小剂量链脲佐菌素建立糖尿病大鼠模型;以糖尿病建模成功的大鼠与16只正常大鼠以结扎冠状动脉前降支制作急性心肌梗死模型;另外16只大鼠只开胸不造模、不给药作为假手术组。心肌梗死造模术后2周,处死各组动物,取左心室梗死区及相邻区域心肌,采用免疫组化方法检测各组大鼠缺血周边区微血管密度（MVD）,Western blotting法检测血管内皮生长因子（VEGF）及其信号转导蛋白[磷酸化蛋白激酶B（p-Akt）、内皮型一氧化氮合酶（eNOS）、磷酸化eNOS（p-eNOS）]、内皮抑素蛋白的表达。组间数据比较采用单因素方差分析,样本均数间两两比较采用q检验,两组计量资料比较采用t检验。结果3组成活大鼠分别为：糖尿病心肌梗死组12只,非糖尿病心肌梗死组（对照组）13只,假手术组14只。与对照组相比,糖尿病组缺血心肌新生血管明显减少（19．7±3．8比14．2±3．6,q=2．98,P〈0．05）。糖尿病组、假手术组VEGF表达显著低于对照组（分别为0．89±0．12、0．65±0．23和1．53±0．20,F=6．52,P〈0．01）。糖尿病组和对照组p-Akt和eNOS蛋白表达差异无统计学意义（t值分别为3．02、2．78,P〉0．05）,但糖尿病组p-eNOS蛋白表达显著减少（0．49±0．09比1．16±0．12,t=5．68,P〈0．05）。糖尿病组内皮抑素表达显著高于对照组（4．6±0．6比2．3±0．4,t=8．63,P〈0．05）。结论糖尿病大鼠急性缺血心肌的新生血管生成低下,VEGF转导通路在多个水平上参与其中,并与内皮抑素共同作用。     

西咪替丁治疗大鼠非乙醇性脂肪性肝炎

目的:探讨西咪替丁对大鼠非乙醇性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)的治疗作用及其可能的作用机制．方法:将30只♂SD大鼠随机分为对照组(n= 10)、模型组(n=10)和治疗组(n=10),模型组和治疗组给予高脂饮食喂养,治疗组给予西咪替T200 mg／(kg·d)灌胃,12 wk后处死大鼠取血测定ALT、AST、血糖、胰岛素和透明质酸含量,观察肝脏组织学变化并测定肝组织CYP450及CYP2E1含量．光镜下观察肝组织病理学变化,进行肝组织炎症活动计分．结果:与对照组比较,模型组大鼠ALT、AST、血糖、胰岛素和透明质酸水平以及CYP450和CYP2E1含量升高,而治疗组ALT (t=5．16,P<0．05),AST(t=5．45,P<0．05), CYP450(t=3．91,P<0．05),CYP2E1(t=1．95, P<0．05),肝湿质量(t=9．24,P<0．05)及透明质酸(T=55,P<0．05)均显著低于模型组,治疗组肝组织炎症活动度计分显著低于模型组(6．40±5．85 vs 7．80±4．60,t=2．59,P<0．05),血糖、胰岛素与模型组比较无显著差异(P>0．05)．结论:西咪替丁能显著减轻NASH大鼠肝脏功能及形态损伤,可能是通过抑制肝细胞微粒体CYP450和CYP2E1的表达发挥作用．     

血管内皮生长因子和内抑素在大鼠糖尿病肾病发病中的作用

目的研究血管内皮细胞生长因子（VEGF）作为促血管生长因子和内抑素（ENS）作为血管生成抑制因子,在糖尿病肾病（DN）血管生成中的作用。方法以正常大鼠为对照,30只雄性Wistar大鼠建成糖尿病模型,分别于糖尿病模型建立2、4、8周观察24小时尿白蛋白排泄率（UAER）,用免疫组织化学染色显示大鼠肾组织VEGF和ENS的表达及半定量分析;用酶联免疫法测定血中VEGF和ENS的浓度,根据DN病理学改变确定DN的血管增生严重性,分析内皮细胞增生、肾小球中弥漫或结节病变及肾小球硬化与VEGF和ENS的关系;采用RT-PCR的方法观察大鼠肾脏VEGF和ENSmRNA的表达。结果DN大鼠肾组织VEGF和ENS的mRNA和蛋白表达水平明显高于对照组（P〈0．05）;DN大鼠血清VEGF和ENS水平也明显高于对照组（P〈0．05）。DN大鼠UAER与血浆VEGF和ENS相关（r=0．468,P〈0．01;r=0．395,P〈0．05）,DN大鼠无论在血中还是肾组织免疫组化和RT-PCR都有VEGF和ENS的表达,VEGF的表达与DN血管增生程度密切相关（r=0．404～0．476,P〈0．01～0．05）,ENS的表达也与DN严重程度密切相关（r=0．409~0．617,P〈0．05～0．01）,血浆VEGF和ENS二者具有正相关性（r=0．484,P〈0．01）。结论VEGF和ENS与DN血管增生密切相关。     

老年高血压的组织因子以及血管内皮生长因子变化

目的了解老年原发性高血压患者冠状动脉粥样斑块及肱动脉内皮功能与组织因子和血管内皮生长因子的关系。方法老年原发性高血压患者175例,男96例,女79例,年龄(73±17)岁。应用B超扫查肱动脉,测量在静息和反应性充血时肱动脉内径,用冠状动脉造影了解冠状动脉狭窄程度。用酶联免疫吸附法测定血组织因子和血管内皮生长因子水平。分析冠状动脉造影及超声结果与组织因子和血管内皮生长因子水平的关系。结果①高血压分级与组织因子和血管内皮生长因子水平与冠状动脉狭窄分级呈正相关,与内皮依赖性血管扩张负相关;②随着病变冠状动脉支数增加,组织因子和血管内皮生长因子水平亦增多;③组织因子与收缩压呈直线相关(r=0．483,P<0．001)、血管内皮生长因子与收缩压呈直线相关(r=0．426,P<0．001);Flow-MD与组织因子呈负相关(r=-0．613,P<0．001),与血管内皮生长因子呈负相关(r=-0．679,P<0．001)与收缩压呈负相关(r=-0．661,P<0．001);组织因子、血管内皮生长因子及Flow-MD与舒张压、平均压、年龄、性别及吸烟之间无明显相关。结论老年原发性高血压患者血液中组织因子、血管内皮生长因子水平增高与高血压分级、肱动脉内皮细胞功能受损及冠状动脉粥样硬化关系密切。     

实验性肝纤维化大鼠肝脏中TIMP-2基因及蛋白表达阻断研究

目的:探讨以TIMP-2为靶基因,应用反义寡核苷酸 (asON)技术抑制其在肝组织中的表达对大鼠肝纤维化发展的影响．方法:22只大鼠分为治疗组(n=6)、模型组(n=6)和正常对照组(n=10)．以人血白蛋白免疫攻击方法制备免疫诱导型肝纤维化大鼠模型．模型制备过程中,治疗组大鼠以尾静脉注射方式给予针对TIMP-2的硫代反义寡核苷酸．RT-PCR、原位杂交、免疫组化、ELISA 等方法检测TIMP-2的转录以及表达水平．用病理学检查以及Ⅰ、Ⅳ型胶原的免疫组化结果分析肝纤维化发展程度．通过胶原纤维特殊染色及电镜等观察asON对大鼠肝纤维化的影响．结果:治疗组病理学分级状况优于模型组(u=2．071, P<0．05)．治疗组血清TIMP-2低于模型组(T=55, P<0．05),高于正常对照组(T=55,P<0．05)．治疗组肝组织TIMP-2 mRNA表达弱于模型组(t=3．332,P<0．05), 高于正常对照组(t=5．550,P<0．05)．Ⅳ型胶原免疫组化图像定量分析结果显示,治疗组与模型组有显著差异(t =2．310,P<0．05),其表达较低,但仍高于正常对照组(t= 3．623,P<0．05)．治疗组Ⅰ型胶原免疫组化图像扫描结果与模型组相比,其数值亦较低(t=2．845,P<0．05)．治疗组肝窦基底膜胶原沉积较轻,与模型组无明显差异．结论:抑制TIMP-2在肝组织中的表达可以减缓大鼠肝纤维化发展．     

普罗布考对急性冠状动脉综合征患者血管内皮损伤的干预效应

目的 观察普罗布考早期治疗对急性冠状动脉综合征（ACS）患者的血管内皮功能的影响。方法 49例ACS患者随机分为两组,普罗布考组（P组,n=24）和常规治疗组（C组,n=25）,检测外周血氧化低密度脂蛋白（ox-LDL）、一氧化氮（NO）及循环内皮细胞（CEC）数量,应用高频超声测肱动脉内皮依赖血管舒张功能（FMD）,用药治疗3个月,观察治疗前后指标变化。结果 P组患者治疗后与治疗前相比,外周血中ox-LDL和CEC水平明显降低（P〈0．01）,NO含量明显升高（P〈0．01）,FMD明显升高（P〈0．05）。P组患者治疗后与C组相比,外周血中ox-LDL和CEC水平明显降低（P〈0．01,P〈0．05）,NO明显升高（P〈0．01）,FMD明显升高（P〈0．05）。C组CEC、NO含量及FMD含量治疗前后差异无统计学意义（P〉0．05）。P组治疗后血浆ox-LDL与CEC呈正相关（r=0．385,P〈0．01）,与NOFMD呈负相关（r=-0．517,P〈0．01）。结论 普罗布考具有抗氧化、降低ox-LDL,提高ACS患者血管内皮功能的作用。     

Paracrine role of adventitial superoxide anion in mediating spontaneous tone of the isolated rat aorta in angiotensin II-induced hypertension.

The relationship between vascular generation of superoxide anion and spontaneous tone observed in the isolated aorta was studied in hypertensive rats infused with angiotensin II. Aortic rings from hypertensive, but not from sham-operated rats, demonstrated oscillatory spontaneous tone that represented 52+/-5.6% of the maximal contraction to KCl. Spontaneous tone was prevented by calcium-free buffer or by blocking calcium influx through L-type calcium channels with nifedipine. The production of superoxide anion measured by lucigenin chemiluminescence was up to 15-fold higher than in sham-operated rat aorta. The adventitial site of production of superoxide anion was suggested by the fact that lucigenin chemiluminescence was 5.5-fold higher from the adventitia than from the intima. This was confirmed histochemically by demonstrating that the adventitia was the site of reduction of nitroblue tetrazolium as well as immunohistochemical staining of NAD(P)H oxidase subunit proteins. A causal link between superoxide anion production by NAD(P)H oxidase and the spontaneous tone is suggested by the fact that superoxide dismutase or the inhibitor of NAD(P)H oxidase, diphenylene iodonium, decreased both superoxide anion production and spontaneous tone. L-NAME or removal of the endothelium from the aorta had no significant effect on superoxide anion levels or spontaneous tone. However, although superoxide dismutase decreased superoxide anion levels in the presence of L-NAME or in endothelium-denuded rings, it no longer inhibited the tone. This suggests that the effect on tone of superoxide anion originating in the adventitia is mediated by inactivating endothelium-derived nitric oxide, which promotes smooth muscle calcium influx and spontaneous tone. The adventitia is not a passive bystander during the development of hypertension, but rather it may have an important role in the regulation of smooth muscle tone.     

冠心病血管内皮功能、颈动脉硬化与冠脉病变的关系

目的：观察冠心病（CAD）患者内皮依赖性舒张功能及颈动脉粥样硬化的状况及与冠状动脉病变的相关性。方法：选经冠状动脉造影确诊为CAD的患者106例（观察组），冠状动脉造影证实无冠状动脉狭窄的健康者40例（正常对照组），采用二维超声检测肱动脉内皮依赖性和非依赖性舒张功能及颈动脉粥样硬化病变。颈动脉粥样硬化斑块积分采用Sutton法，并与冠状动脉病变程度进行对比分析。结果：CAD组内皮依赖性血管舒缩功能（FMD）比正常对照组明显降低（P〈0．01），内皮非依赖性血管舒缩功能与正常对照组比较无显著性差异（P〉0．05）；CAD组颈动脉内膜中层厚度（IMT），斑块积分显著高于正常对照组（P〈0．01）。FMD与冠脉病变程度呈负相关（r=-0．651，P〈0．001），颈动脉IMI及斑块积分与冠脉病变程度呈正相关（r=0．871，0．702，P〈0．001）。结论：内皮细胞功能障碍和颈动脉粥样硬化与冠状动脉粥样硬化的病变相平行。     

Absence of role of endothelium in the response of isolated porcine coronary arteries to acetylcholine

Isolated precontracted arteries of various vascular beds relax in response to acetylcholine only if the endothelium is present. One explanation for this is that this drug stimulates the endothelial cells to release a vasodilator substance that in turn relaxes the underlying smooth muscle. To determine whether this mechanism is concerned also in the acetylcholine contraction of isolated porcine coronary arteries transverse strips of the extramural part of the left circumflex artery were used for recording isometric tension in the muscle bath. Dose-response curves for acetylcholine showed no significant difference before and after removal of endothelium. As a functional check on the removal of endothelium, the responsiveness of each preparation to a known endothelium dependent dilator (substance P) was tested before and after removal. These findings suggest that endothelial cells are not concerned in the acetylcholine induced contraction of porcine coronary arteries. Acetylcholine appears to act directly on smooth muscle of the porcine artery.     

Increased superoxide generation is associated with pulmonary hypertension in fetal lambs: a role for NADPH oxidase

Ligation of the ductus arteriosus in utero produces pulmonary hypertension and vascular remodeling in fetal and newborn lambs. However, the mechanisms producing these vascular changes are not well defined. Because reactive oxygen species (ROS) have been implicated as mediators of smooth muscle cell proliferation, we hypothesized that increased formation of ROS may be involved in the pathophysiology of pulmonary hypertension after in utero ductal ligation. Using ethidium fluorescence, we demonstrated an increase in superoxide levels after 9 days of ductal ligation compared with control lungs (P<0.05) that was localized to the adventitia and smooth muscle cells of hypertensive vessels. SOD-1 and SOD-2 protein levels and activities in lung, vein, and artery of hypertensive lambs were unchanged relative to controls after 2 days of ductal ligation. However, after 9 days, superoxide dismutase (SOD) activity was significantly decreased in arteries from ligated lambs without associated changes in SOD protein expression (P<0.05). Examination of NADPH oxidase expression as a potential source of the superoxide production indicated that the levels of p67phox, a subunit of the NADPH oxidase complex, were significantly increased in the pulmonary arteries, but not veins, from the ligated lung as early as 2 days (P<0.05). Functional analyses demonstrated that reducing superoxide levels significantly increased the NO-mediated relaxation of pulmonary arteries isolated after 9 days, but not 2 days, of ductal ligation (P<0.05). These results suggest that increased NADPH oxidase expression may increase levels of superoxide in persistent pulmonary hypertension of the newborn lung tissue, and that increased superoxide blunts vascular relaxations to exogenous NO while stimulating smooth muscle cell growth.     

Responses of the rabbit epicardial coronary artery to acetylcholine and adrenoceptor agonists.

STUDY OBJECTIVE--The aim was to identify the role of the endothelium in mediating the responses to acetylcholine in the rabbit coronary artery, and to determine whether alpha or beta adrenergic stimulation may cause relaxation via endothelial receptors in the coronary arteries of this species. DESIGN--Responses to acetylcholine and adrenoceptor agonists were compared in isolated ring preparations with and without endothelium. The adrenoceptor agonists were examined in the presence of phentolamine or propranolol to block alpha and beta adrenoceptors, respectively. EXPERIMENTAL MATERIAL--30 New Zealand white rabbits (2.3-3.4 kg) were killed by an overdose of barbiturate and exsanguination, and the left epicardial coronary artery was dissected free. Ring preparations were suspended in organ baths under isometric tension and, where required, the tone of the preparations was raised by KC1. MEASUREMENTS AND MAIN RESULTS--Concentrations of acetylcholine up to 10(-6) mol.litre-1 produced dose dependent relaxation of the preparations with endothelium intact, but no relaxation in preparations denuded of endothelium. At higher concentrations, a marked vasoconstrictor response was seen in all preparations regardless of the presence of endothelium. At basal tone, acetylcholine produced vasoconstriction which reached a maximum of 1.0 (SEM 0.14)g tension in preparations with endothelium and 1.74(0.27) g tension in those without endothelium (p less than 0.05). In coronary arteries pretreated with 50 mumol.litre-1 phenoxybenzamine to block alpha adrenoceptors, noradrenaline, isoprenaline, and salbutamol produced dose dependent relaxation of the preparations; this was unaffected by the absence of endothelium. In vessels not pretreated with phenoxybenzamine, propranolol inhibited the relaxation to noradrenaline and isoprenaline but again there was no difference between vessels with and without endothelium. CONCLUSIONS--In the rabbit isolated epicardial coronary artery, acetylcholine produces an endothelium dependent relaxant response over a limited concentration range; a vasoconstrictor response via smooth muscle dominates at higher concentrations. beta Adrenoceptors mediating relaxation are present on the smooth muscle, but there was no evidence for either alpha or beta adrenoceptor mediated responses via the endothelium. Important differences with coronary arteries from other species are discussed.     

Intimal estrogen receptor (ER)beta, but not ERalpha expression, is correlated with coronary calcification and atherosclerosis in pre- and postmenopausal women

BACKGROUND: Controversy exists over the association of estrogen and cardiovascular disease. Estrogen receptors (ERs) alpha and beta are expressed in the endothelial cells and vascular smooth muscle cells (VSMCs) of many arteries, but the relative importance of ERalpha or ERbeta in mediating the vascular response to estrogens is not well defined, particularly in humans. We have shown previously that postmenopausal women receiving hormone therapy (HT) had lower mean coronary artery calcium, plaque area, and calcium-to-plaque ratio compared with untreated women. In this study, we examined coronary artery ERalpha and ERbeta expression in pre- and postmenopausal women as a function of plaque area, calcium area, calcium-to-plaque ratio, and estrogen status. METHODS: Coronary arteries were obtained at autopsy from a total of 55 women: nine premenopausal women, 13 postmenopausal women on HT and 33 untreated postmenopausal women (non-HT). Coronary calcification was quantified by contact microradiography, and atherosclerotic plaque area was measured histologically. Coronary artery cross-sections were immunostained for ERalpha and ERbeta, and the amount of receptors was estimated semiquantitatively in each arterial wall layer (intima, adventitia, and media). Double immunofluorescence was used to colocalize ERalpha and ERbeta with smooth muscle actin, a marker of VSMCs. RESULTS: ERbeta and ERalpha were expressed in all artery wall layers, but most avidly in the media (P = 0.001), and colocalized with VSMCs. ERbeta expression exceeded ERalpha expression in all wall layers (P < 0.001) and was adjacent to areas of calcium deposition. ERbeta expression in the intimal layer correlated with calcium content, plaque area, and calcium-to-plaque ratio (all P < 0.01) and tended to be greater in non-HT than in HT women (P = 0.06). ERalpha expression did not vary significantly among groups, nor did it correlate with calcium content, plaque area or calcium-to-plaque ratio. Expression of ERalpha but not ERbeta declined with age (P < 0.01) in HT women only. Age had no effect on ERalpha or ERbeta expression in non-HT or premenopausal women. CONCLUSIONS: ERbeta is the predominant ER in human coronary arteries and correlates with coronary calcification, a marker of severe atherosclerosis. Increased ERbeta expression is linked to advanced atherosclerosis and calcification independent of age or hormone status. Future pharmacogenetic studies that target this receptor are needed to confirm causality.     

Correlating androgen and estrogen steroid receptor expression with coronary calcification and atherosclerosis in men without known coronary artery disease

BACKGROUND: Accumulating data emphasize the gender specificity of key components of the atherosclerotic process and the importance of gonadal steroids on the human vasculature. Steroid receptors, including the androgen receptor (AR) and estrogen receptors (ERs) alpha and beta are expressed in key vascular tissues, including endothelial cells and vascular smooth muscle cells. However, the relative abundance and importance of these receptors in the coronary artery are not well defined, particularly in men. We therefore examined AR, ER alpha, and ER beta expression as a function of key components of atherosclerosis, namely plaque and calcium area, in male human coronary arteries. METHODS: Coronary arteries were obtained at autopsy from 24 men without known coronary artery disease. Coronary calcification was measured by contact microradiography, and atherosclerotic plaque area was quantified histologically. Coronary artery cross-sections were immunostained for AR, ER alpha, and ER beta and then measured semiquantitatively in each arterial wall layer (intima, adventitia, and media). RESULTS: AR, ER beta, and ER alpha were expressed in all artery wall layers but most avidly in the media (P < 0.001). ER beta exceeded ER alpha expression (P < 0.0005). AR expression in the media correlated negatively with plaque area (P = 0.006, R = -0.55), whereas intimal ER beta expression correlated positively with plaque area (P = 0.012, R = 0.50). CONCLUSIONS: We conclude that both AR and ER beta are important in relatively early coronary atherosclerosis, but inversely so, because decreasing AR and increasing ER beta expression correlate with more extensive atherosclerosis. ER beta seems to be the predominate ER in coronary arteries harvested from men without known coronary artery disease. Interventional studies are required to assess the functional significance of these observations.     

Expression of glutaredoxin in human coronary arteries: its potential role in antioxidant protection against atherosclerosis

Oxidative stress is considered an important factor in atherogenesis. Mammalian cells have a complex network of antioxidants such as catalase, superoxide dismutase, and glutathione peroxidase. However, the mechanisms that regulate the cellular redox state in the vessel wall remain unclear. Recent study has shown that thioredoxin, a thiol-disulfide oxidoreductase, is expressed in atherosclerotic plaques of human carotid arteries. In this study, we investigated the localization and expressional change of glutaredoxin and thioredoxin, two important members of the thiol-disulfide oxidoreductases, in autopsy samples of human coronary arteries. In nonatherosclerotic coronary arteries, glutaredoxin was expressed in endothelial cells, in fibroblasts of the adventitia, and most intensely in medial smooth muscle cells. Interestingly, in atherosclerotic lesions such as hypercellular lesions, the infiltrating macrophages highly expressed glutaredoxin. The expressional pattern of thioredoxin was quite similar to that of glutaredoxin. Western blot analysis demonstrated that hydrogen peroxide stimulated the expression of glutaredoxin in a time- and dose-dependent manner in cultured human coronary artery smooth muscle cells. Fluorescence microtopography with dihydroethidium demonstrated that the generation of reactive oxygen species was associated with the expression of glutaredoxin. These results suggest the possible involvement of thiol-disulfide oxidoreductases in antioxidant protection in human coronary arteries.     

Inhibition of coronary artery superoxide dismutase attenuates endothelium-dependent and -independent nitrovasodilator relaxation.

Isolated bovine coronary arteries were treated with 10 mM diethyldithiocarbamate (DETCA) for 30 minutes to deplete the cytosolic ZnCu form of superoxide dismutase (SOD). This treatment completely inhibited the endothelium- and cGMP-dependent relaxation to acetylcholine (mediated via the endothelium-derived relaxing factor, which is thought to be nitric oxide) without significantly inhibiting endothelium-dependent relaxation to arachidonic acid (mediated by prostaglandins). DETCA treatment of endothelial cells cultured from the coronary arteries inhibited bradykinin-elicited release of endothelium-derived relaxing factor, which was detected by bioassay on an isolated rabbit aorta in the presence of extracellular SOD. DETCA also inhibited cGMP-associated relaxations to nitric oxide and to vasodilators thought to function via the generation of this mediator (nitroglycerin and nitroprusside), but cAMP-associated relaxations to isoproterenol and papaverine were not altered. The inhibitory effects of DETCA against the relaxation to nitroprusside and nitroglycerin were attenuated by severe hypoxia. DETCA treatment of isolated coronary arterial smooth muscle or cultured endothelial cells produced an increase of chemiluminescence elicited in the presence of lucigenin, a detector of superoxide anion generation. The addition of SOD markedly attenuated the effects of DETCA treatment on arterial relaxation and chemiluminescence. Therefore, control of cellular superoxide anion levels by endogenous SOD appears needed for the release of endothelium-derived relaxing factor and relaxation of vascular smooth muscle to nitrovasodilators mediated via cGMP in the bovine coronary artery, but SOD is not critical for other endothelium-dependent or cAMP-associated relaxant mechanisms.