Immunohistochemical analysis of CD31 expression in myocardial tissues from autopsies of patients with ischemic heart disease

CD31, a transmembrane protein expressed on endothelial and hematopoietic cells, plays important roles in leukocyte trafficking, mechanotransduction, angiogenesis, vascular permeability, and regulation of cellular responsiveness. CD31 immunoreactivity is employed as a sensitive and specific endothelial marker in diagnostic pathology. In this study, CD31 expression in myocardial tissues from deceased patients with ischemic heart disease and a mouse model of acute myocardial infarction were examined by immunohistochemical staining. We examined 24 neutral formalin-fixed, paraffin-embedded myocardial tissue samples obtained within 48 h postmortem from the autopsies of patients who were diagnosed with ischemic heart disease. CD31 expression was observed in vascular endothelial and endocardial cells. In necrotic myocardium, diffusion of CD31 antigen was observed. Elevated CD31 expression was observed around myocardial cells undergoing remodeling, suggesting that endothelial proliferation occurred at these sites. In contrast, fibrotic myocardial foci did not show upregulated CD31 expression. The same CD31 expression characteristics as those observed in the human samples were observed in the mouse model. CD31 immunostaining as an endothelial and microvasculature marker may be a useful complement to conventional staining techniques currently used in the diagnosis of ischemic heart disease, and may allow the timing and process of myocardial remodeling to be analyzed in detail.     

Immunohistochemical analysis of thrombomodulin expression in myocardial tissue from autopsy cases of ischemic heart disease

Thrombomodulin is a transmembrane glycoprotein that is ubiquitously expressed on the surface of vascular endothelial cells. Thrombomodulin exerts its anticoagulant effects by combining with thrombin, activating protein C, and inactivating the coagulation factors FVa and FVIIIa. Clinically, thrombomodulin is also known as a marker of vascular injury because it circulates freely in response to endothelial injury. In this study, myocardial tissue from cases of ischemic heart disease was subjected to immunohistochemistry by thrombomodulin. We examined 40 neutral-formalin-fixed, paraffin-embedded myocardial tissue samples from autopsy cases that were diagnosed with ischemic heart disease (within 48 h postmortem). Thrombomodulin expression was observed in vascular endothelial cells between myocardial cells and in mesothelial cells of the epicardium. In necrotic myocardium, diffusion of thrombomodulin, which reflected endothelial injury, was observed. Upregulated thrombomodulin expression was observed around myocardial cells under ongoing remodeling, which suggested endothelial proliferation in these locations. Completed fibrotic foci of the myocardium did not show upregulated thrombomodulin expression. In a mouse model of acute myocardial infarction, the same phenomena as that found in human samples were observed by immunohistochemistry of thrombomodulin. Immunostaining of thrombomodulin, as a marker for endothelial injury or myocardial remodeling, may be useful for supplementing conventional staining techniques in the diagnosis of ischemic heart disease in forensic pathology.     

Immunohistochemical analysis of vimentin expression in myocardial tissue from autopsy cases of ischemic heart disease

Vimentin is a type III intermediate filament cytoskeletal protein that is expressed mainly in cells of mesenchymal origin and is involved in a plethora of cellular functions. In this study, myocardial tissues from patients with ischemic heart disease and a mouse model of acute myocardial infarction were subjected to immunohistochemistry for vimentin. We first examined 26 neutral formalin-fixed, paraffin-embedded myocardial tissue samples from autopsies of patients that were diagnosed with ischemic heart disease within 48 h postmortem. Myocardial cells were negative for vimentin, whereas non-myocardial cells, including vascular endothelium, vascular smooth muscle, fibroblasts, nerve fibers, adipocytes and mesothelial cells, showed positivity. Elevated vimentin expression was observed around myocardial cells undergoing remodeling, suggesting fibroblastic and endothelial proliferation in these locations. By contrast, myocardial foci that were completely fibrotic did not show upregulated vimentin expression. Inflammatory foci including macrophages and neutrophils were clearly visualized with vimentin immunostaining. The same vimentin expression phenomena as those found in human samples were observed in the mouse model. Our study indicates that immunostaining of vimentin as a marker for myocardial remodeling and the dynamics of all non-myocardial cell types may be useful for supplementing conventional staining techniques currently used in the diagnosis of ischemic heart disease.     

Myocardial CD36 expression and fatty acid accumulation in patients with type I and II CD36 deficiency

Long-chain fatty acids (LCFA) are one of the major cardiac energy substrates, so understanding LCFA metabolism may help in elucidating the mechanisms of various heart diseases. CD36 is a multifunctional membrane glycoprotein that acts not only as a receptor for thrombospondin, collagen and oxidized low density lipoprotein but also as a receptor for LCFA. We investigated the relationship between CD36 expression in myocardial capillary endothelial cells and myocardial LCFA uptake in patients with CD36 deficiency. We analyzed CD36 expression in blood cells from 250 patients with heart diseases by means of a flow cytometer. In 218 patients, myocardial LCFA scintigraphy was performed with¹²³I-β-methyl-p-iodophenyl pentadecanoic acid (BMIPP). In 5 patients, myocardial capillary endothelial cells were examined immunohistochemically for CD36 expression. Eleven patients (4%) showed signs of type I CD36 deficiency (neither platelets nor monocytes expressed CD36). Twenty patients (8%) had type II CD36 deficiency (monocytes expressed CD36 but platelets did not). In all 11 patients with type I CD36 deficiency, no BMIPP accumulation was observed in the heart, but in 13 patients with type II CD36 deficiency, BMIPP accumulation in the heart was focally reduced, but there were no patients without BMIPP accumulation in the heart. Although the myocardial capillary endothelial cells from two CD36-positive patients expressed CD36, those from two patients with type I CD36 deficiency did not. In a patient with type II CD36 deficiency, some capillary endothelial cells displayed patchy CD36 expression. CD36 deficiency was documented in 31 (12%) patients with heart diseases. Because CD36 was not expressed in the myocardial capillary endothelial cells in patients with type I CD36 deficiency, type I CD36 deficiency is closely related to lack of myocardial LCFA accumulation and metabolism in the myocardium.     

基质金属蛋白酶参与左心室重构的机制探讨

目的 探讨基质金属蛋白酶家族（MMPs）MMP-2、3、9与心衰患者心肌重构的关系。方法 选择因二尖瓣关闭不全，心脏病接受二尖瓣置换术的CHF病人39例，正常对照38例（其中8例来自意外伤亡的器官捐献者）。彩色多普勒超声心动图检测心功能参数，免疫沉淀法检测心肌组织MMP-2、3、9。结果 瓣膜病所致心力衰竭病人心肌组织呈心肌重构的病理改变；心衰病人心肌组织蛋白表达较正常人高，且随心功能的恶化，MMP-2、3、9含量相对越高（P〈0．05或0．01）。结论 MMP-2、3、9表达量的增高与心肌重构的病理过程。是影响心衰患者心功能的重要因素之一。     

Increased Deposition of von Willebrand Factor in the Rat Heart after Local Ionizing Irradiation

BACKGROUND AND PURPOSE: Von Willebrand factor (vWf), a glycoprotein involved in blood coagulation, is synthesized by endothelial cells. Increased amounts of vWf in blood plasma or tissue samples are indicative of damaged endothelium. In the present study, mRNA expression and localization of vWf were determined in irradiated rat heart tissue. MATERIAL AND METHODS: Sprague-Dawley rats received local heart irradiation with a single dose of 0, 15, or 20 Gy. Hearts were dissected at different time points (up to 16 months) after irradiation. In a second experiment, rats were injected with the radioprotector amifostine (160 mg/kg, i. p.) 15-20 min before irradiation and sacrificed after 6 months. Immunohistochemistry was performed using a polyclonal anti-vWf antibody. Serial sections were subjected to a general rat endothelial cell immunostaining (RECA-1) or a collagen staining (picrosirius red). mRNA expression was determined by using PCR. RESULTS: In control tissue, all endothelial cells lining the lumen of the endocardium and coronary arteries, but not capillary endothelial cells, were stained for vWf. 1 month after irradiation with both 15 and 20 Gy, myocardial capillaries became immunoreactive. From 3 months onward, staining was observed also within the extracellular matrix (ECM) of fibrotic areas. At mRNA level, no changes in vWf could be observed at all time points after irradiation, suggesting that vWf deposition was not due to increased biosynthesis of the protein. In sections of amifostine-treated rat hearts, vWf staining was increased to a lesser extent. CONCLUSION: These dose- and time-dependent increases in deposition of vWf indicate the presence of damaged endothelium in the irradiated rat heart. These increases in vWf accumulation precede development of fibrosis in the subendocardial layer and myocardium of the left ventricles, right ventricles, and atria.     

猪缺血心肌内膜注射VEGF基因的长期疗效观察

目的探讨经心内膜心肌内直接注射血管内皮生长因子(VEGF)基因治疗猪心肌缺血后的远期心脏电机械活动及心功能改善情况。方法30只实验用小香猪随机均分为对照组(n=15)和治疗组(n=15)。建立心肌缺血模型后,通过NOGA系统经心内膜分别将空载质粒pIRES2-EGFP及质粒pIRES2-EGFP-hVEGF165直接注射至对照组或治疗组缺血部位心肌内。在注射前及注射后1年,分别应用左心室电机械标测(LVEMM)监测局段线性缩短率(LLS)和单极电压(UpV),并用超声监测M型局部室壁运动幅度和背向散射积分的心动周期变异(CVIB)、左心室射血分数(LVEF)。1年后处死动物,组织学切片观察心肌组织中毛细血管生成情况。结果LVEMM监测显示,注射后1年治疗组LLS显著高于注射前及对照组(P<0.05),而UpV与注射前及对照组比较无显著差异(P>0.05);超声监测显示,治疗组注射后1年的局部室壁运动幅度和CVIB、LVEF均显著高于对照组(P<0.05)。组织学检查显示,治疗组心肌内毛细血管数目(38.1±4.8/HP)显著高于对照组(13.2±5.1/HP,P<0.01)。结论经心内膜直接注射pIRES2-...     

自发性高血压大鼠心肌血管内皮生长因子表达水平的检测

 目的探讨血管内皮生长因子(Vascular endothelial growth factor,VEGF)在自发性高血压大鼠(Spontaneously hyper-tensive rat,SHR)心肌细胞中的表达情况,分析其与高血压时心肌毛细血管稀少及微小动脉"重塑"(remodeling)的关系.方法应用免疫组化SP染色法及计算机图像分析技术,对幼年(6周,n=15)和成年(12个月,n=15)SHR大鼠及幼年(6周,n=10)和成年(12个月,n=10)同系正常血压对照组京都Wistar大鼠(Wistar-Kyoto,WKY)心肌VEGF蛋白的表达水平进行了定量分析.结果在4组大鼠心脏各部(心房肌、心室肌、房间隔、室间隔等)心肌细胞浆中及冠状动脉各级分支的血管平滑肌细胞中均见有阳性VEGF表达,6周龄、12个月龄WKY及6周龄SHR三组间表达水平差异无统计学意义(P＞0.05),而12个月龄SHR组表达水平较其他三组均高(P＜0.05).结论随着成年SHR大鼠血压的升高,心肌及血管平滑肌细胞中VEGF蛋白的表达水平上调,可能是对高血压所致的靶器官继发性毛细血管减少的一种代偿反应,高表达的VEGF可对抗由于缺氧诱导的内皮细胞凋亡,维持其存活,并促进微血管再生.     

Analysis of apical remodeling in gated myocardial perfusion SPECT imaging in ischemic cardiomyopathy

Background  A divergent pattern (DP) of the left ventricle (LV) is an expression of apical remodeling in myocardial perfusion gated single-photon emission computed tomography (SPECT) of patients with ischemic cardiomyopathy (ICM). Methods and Results  We consecutively studied 156 patients (mean age, 63 years; 24 women) with ICM (LV ejection fraction, ≤40%) using gated SPECT and technetium-labeled agents. Apical remodeling was considered to exist when a DP was observed. Apical remodeling was noted in 30% of patients, all of whom had a history of anterior myocardial infarction. A divergent pattern was observed more frequently in younger patients and in those with ST-segment elevation on their electrocardiograms. The longer the interval between the infarction and the performance of gated SPECT, the more prevalent were the LV dilatation and DP. A divergent pattern was associated with cardiac death and heart failure only in patients with scintigraphic criteria for myocardial viability. Conclusions  A divergent pattern in gated SPECT, as an expression of apical remodeling, can be observed in up to a third of patients with ICM, all with a history of anterior infarction. The longer the time between the infarction and the gated SPECT, the more prevalent the LV dilatation becomes. Apical remodeling is a variable predicts mortality in patients with scintigraphic criteria for viability. This study was partially funded by grants from Fundación Carolina (Madrid, Spain) and the Redes Temáticas de Investigación Cooperativa. Instituto Carlos III (Red C03/01, red temática de entermedades cardiovasculares [RECAVA] (Madrid, Spain).     

宝石CT心肌灌注成像在缺血性心脏病动物模型中的应用

目的对不同缺血时间缺血性心脏病动物模型行CT心肌灌注扫描,评价其在诊断缺血性心脏病中的价值。材料与方法健康新西兰大白兔20只,结扎左前降支近段建立缺血性心脏病模型。于建模后6h、3天、7天、14天行CT首过心肌灌注扫描,扫描结束后取病理,行HE染色和CD31免疫组织化学检查。分析比较各组的血流灌注量(BF)、血容量(BV)、平均通过时间(MTT)之间的差异及各指标与微血管密度(MVD)间的关系。结果 (1)缺血心肌BF值随缺血时间延长其值呈下降趋势,各不同缺血时间组BF值差异有统计学意义(P<0.05);(2)缺血心肌BF值与MVD之间存在负相关rp=-0.525,P=0.018。结论宝石CT首过心肌灌注可以评估不同缺血时间缺血心肌的灌注特征,有助于定性评价缺血性心脏病。     

Molecular and cellular mechanisms of myocardial remodeling

Summary  Myocardial remodeling is inextricably intertwined with the development and progression of heart failure. It is characterized by alterations in ventricular geometry and function. At the cellular level, ventricular remodeling consists of myocyte hypertrophy, neurohumoral activation, matrix remodeling, altered gene expression, immune system activation, and apoptosis. Many factors, including neurohormones, cytokines, growth factors, oxidative stress, and mechanical strain, can trigger the molecular and cellular events observed in remodeling, thus suggesting that they may play a role in myocardial remodeling. A thorough understanding of the cellular and molecular mechanisms involved in myocardial remodeling may lead to novel pharmacologic or genetic therapies for the treatment and prevention of heart failure.     

Cardiovascular disease among U.S. Navy pilots

This study's objectives were to determine the influence of age and occupational factors on cardiovascular disease (CVD) incidence among U.S. Navy pilots diagnosed with CVD during a 12.5-year time period (n = 150) and to identify precursory diseases associated with CVD. Results showed a relationship between CVD and age; pilots, on the average, were more than 3 years younger at the time of CVD onset than other officers. No occupational factor was associated with CVD; fighter pilots had the highest rates of acute myocardial infarction and chronic ischemic heart disease. Angina pectoris was observed as a precursory disease of chronic ischemic heart disease, and several behaviorally related disorders (e.g., alcoholism) occurred with hypertension. Subsequent research should include all U.S. military pilots to examine, in a larger population, the influence on CVD of such occupational factors as flight in high-performance aircraft. An intervention program should be implemented to modify the lifestyles of pilots who had been hospitalized for hypertension and/or such conditions as obesity and alcoholism.     

Ischemic heart disease

Ischemic heart disease has a number of important manifestations that can be observed on plain films and at fluoroscopy. Special roentgen procedures, such as coronary arteriography and angiocardiography, are extremely useful to the in-depth evaluation of coronary heart disease.

Recognition of morphologic and physiologic abnormalities on plain film and at fluoroscopy can be important in the initial diagnosis of ischemic heart disease, in the follow-up, and in the evaluation of the complications.

Acute congestive heart failure, particularly when associated with a relatively small heart, is one of the hallmarks of acute myocardial infarction. However, marked degrees of cardiomegaly may occur, especially after repeated attacks. A number of lesser manifestations, including myocardial calcification, can be seen. Using relatively simple fluoroscopic techniques, abnormalities of myocardial contraction occur in about 80 per cent of patients and often reflect the severity and extent of existing damage.

The complications of ischemic heart disease range from pulmonary and systemic embolism and infarction through myocardial rupture, aneurysm, and the more rare and esoteric postinfarction and shoulder-hand syndrome.

The role of the informed roentgenologist in the evaluation of a patient suspected of ischemic heart disease has been significant and, with the newer angiographic techniques, is becoming even more important.     

Scintigraphic assessment of the effects of bone marrow-derived mononuclear cell transplantation combined with off-pump coronary artery bypass surgery in patients with ischemic heart disease.

Myocardial SPECT may be useful for assessment of the therapeutic effects and the mechanisms of cardiac regeneration medicine. We aimed to assess first the feasibility and the short-term safety of autologous bone marrow-derived mononuclear cell transplantation (BMCT) into the ischemic myocardium in patients who undergo off-pump coronary artery bypass surgery (OPCAB). In addition, we aimed to assess our hypothesis that the BMCT may help ameliorate myocardial perfusion in patients with ischemic heart disease (IHD) using myocardial perfusion scintigraphy. METHODS: We performed BMCT in 10 patients with IHD during OPCAB. Cells for BMCT were collected by intraoperative bone marrow aspiration or by preoperative cellular apheresis after pretreatment with granulocyte colony-stimulating factor. After OPCAB was performed in all graftable ischemic areas, a total of 3.4 +/- 1.2 x 10(9) mononuclear cells, including 5.2 +/- 1.6 x 10(6) CD34-positive (CD34(+)) cells, were injected into ungraftable ischemic myocardial areas. Dipyridamole-stress and resting (99m)Tc myocardial SPECT was performed before and 1 mo after the procedures. RESULTS: BMCT was performed safely in all patients. Compared with before treatment, myocardial (99m)Tc tracer uptake on the dipyridamole-stress image increased similarly in BMCT- and OPCAB-treated areas, whereas tracer accumulation at rest did not change in all myocardial areas. The improvement of myocardial perfusion was not correlated with the total number of mononuclear cells transplanted. However, it was positively correlated with the number of transplanted CD34(+) cells: (99m)Tc tracer uptake after/before BMCT (ratio) = 1.091 x (CD34(+) cell number [x10(6)])(0.074) (r(2) = 0.48, P < 0.05), although new development of coronary vessels was not documented cineangiographically. Myocardial histopathology in 2 of 3 autopsy cases revealed coronary angiogenesis in the areas corresponding to the sites of BMCT. CONCLUSION: The present study demonstrates the feasibility of BMCT combined with OPCAB. This therapy improves myocardial perfusion possibly via CD34-related development of coronary microvessels.     

Stem cells and cardiovascular disease

Several recent discoveries have shifted the paradigm that there is no potential for myocardial regeneration and have fueled enthusiasm for a new frontier in the treatment of cardiovascular disease-stem cells. Fundamental to this emerging field is the cumulative evidence that adult bone marrow stem cells can differentiate into a wide variety of cell types, including cardiac myocytes and endothelial cells. This phenomenon has been termed stem cell plasticity and is the basis for the explosive recent interest in stem cell-based therapies. Directed to cardiovascular disease, stem cell therapy holds the promise of replacing lost heart muscle and enhancing cardiovascular revascularization. Early evidence of the feasibility of stem cell therapy for cardiovascular disease came from a series of animal experiments demonstrating that adult stem cells could become cardiac muscle cells (myogenesis) and participate in the formation of new blood vessels (angiogenesis and vasculogenesis) in the heart after myocardial infarction. These findings have been rapidly translated to ongoing human trials, but many questions remain. This review focuses on the use of adult bone marrow-derived stem cells for the treatment of ischemic cardiovascular disease and will contrast how far we have come in a short time with how far we still need to go before stem cell therapy becomes routine in cardiovascular medicine.     

MRI of myocardial infarction.

With the advances in magnetic resonance imaging (MRI) technology that have occurred in recent years, it is possible to examine the myocardial status with high spatial and temporal resolutions in the evaluation of ischemic heart disease. The purpose of this article is to review the current status and the role of MRI for the evaluation of myocardial infarction. We discuss the pathophysiology of myocardial infarction, MRI techniques for the evaluation of myocardial status, and the pathophysiological significance of MR signal changes observed in various MRI techniques. We conclude that, with further development of MR techniques and contrast agents, MRI will play an increasing role in the diagnosis of ischemic heart disease. J. Magn. Reson. Imaging 1999;10:686-693.     

高压氧对成年大鼠脑缺血再灌注损伤后脑血管内皮生长因子表达的影响

目的 探讨脑缺血模型大鼠经30 d高压氧(HBO)治疗后大脑血管内皮生长因子(VEGF)表达的变化.方法 24只成年SD大鼠随机分为3组:对照组(C组)8只、缺血再灌注组(IR组)8只、HBO治疗组(HBO组)8只.C组为正常大鼠,IR组和HBO组大鼠经大脑中动脉栓塞(MCAO)1.5 h后再灌注,HBO组行HBO治疗.HBO治疗(0.28 MPa,60 min)30 d,1次/d,于31 d麻醉处死,取脑组织切片,VEGF和CD34免疫组化染色,光镜观察并于损伤区取图,进行相关分析.结果 VECF广泛分布,缺血区表达强烈,3组间有明显差异(P＜0.05),HBO组(13497.14±397.44)低于IR组(22.097.7±616.89);C组未见CD34表达,其他2组集中表达于缺血区;HBO组(5571.16±603.63)表达少于IR组(5349.72±390.88),但差异无统计学意义(P＞0.05).结论 长期HBO治疗抑制大鼠缺血脑组织VEGF的表达,可能对神经细胞具有保护作用,有利于促进脑组织自我修复.     

The sudden and unexpected death of a female-to-male transsexual patient

A 32-year-old woman, who was intramuscularly injected with testosterone enanthate (125 mg) once or twice a month over a two-year period for female-to-male transsexualism, died suddenly. A forensic autopsy was performed to investigate the cause of death. Concentric cardiac hypertrophy was macroscopically observed. In the left and right coronary arteries, atherosclerosis was generally observed within the endothelium. In particular, there was severe stenosis (>90%) at the start of the left descending branch. In the myocardium, both coagulation necrosis and contraction band necrosis were microscopically observed. Moreover, myocardial fibrosis and myocardial calcification were diffusely detected, respectively. The cause of death was diagnosed as ischemic heart disease due to coronary stenosis. There is some debate as to whether cross-hormone replacement is related to the occurrence of coronary artery disease or not, however, it is possible that the development of ischemic heart disease was aggravated by the administration of testosterone enanthate in the current case.     

脐血CD133+细胞移植治疗裸鼠实验性缺血性心肌病

目的探讨人脐血CD133+细胞移植于缺血心肌后的增殖分化情况和对缺血心肌梗塞面积和纤维化程度改善情况.方法从新鲜脐血中纯化的CD133+细胞,PKH26标记后注射于结扎冠状动脉后的裸鼠缺血心肌内,并以无血清培养基注射动物为对照组.2周后观察移植细胞的分化情况,检测心肌纤维化程度和梗塞面积的变化.结果CD133+细胞移植后2周,对照组梗塞面积(26.3±3.8)%,纤维化程度(12.8±1.1)%;而实验组梗塞面积(7.8±1.3)%,纤维化程度(5.5±0.7)%(P<0.05).结论CD133+细胞移植能明显减少实验动物心肌梗塞面积和纤维化程度.