Progression of coronary atherosclerosis relates to the onset of myocardial infarction in an animal model of spontaneous myocardial infarction (WHHLMI rabbits).

Recently, we developed myocardial infarction-prone WHHLMI rabbits from coronary atherosclerosis-prone WHHL rabbits (WHHLCA rabbits) by selective breeding. In this study, we examined the relation of atherosclerotic plaques to the onset of myocardial infarction. We examined myocardial lesions of 378 WHHL rabbits born between 1992 and 2000, and atherosclerosis lesions of 93 WHHLCA and 82 WHHLMI rabbits. The aortic lesions were evaluated as percent surface lesion area. The coronary lesions were evaluated as cross sectional narrowing using sections prepared at 500 or 1,000 microm intervals. Serum lipid levels were assayed with enzymatic methods. The cumulative incidence of fatal myocardial infarction between 11 and 35 months old was 90% in WHHLMI rabbits and 21% in WHHLCA rabbits, respectively. Selective breeding increased the serum cholesterol levels by about 200 mg/dl despite there being no changes in triglyceride levels. Aortic and coronary atherosclerosis progressed markedly in WHHLMI rabbits compared to WHHLCA rabbits. Especially, WHHLMI rabbits over 15 months old showed more than 90% cross sectional narrowing of the left circumflex arteries, main stem of the left coronary artery, and the origin portion of the right coronary artery. In addition, there were no gender differences in atherosclerotic lesions of both aortas and coronary arteries. In conclusion, the present study showed that marked progression of coronary atherosclerosis was probably associated with spontaneous development of myocardial infarction in WHHLMI rabbits.     

Noninvasive detection of macrophages using a nanoparticulate contrast agent for computed tomography

Sudden fibrous cap disruption of 'high-risk' atherosclerotic plaques can trigger the formation of an occlusive thrombus in coronary arteries, causing acute coronary syndromes. High-risk atherosclerotic plaques are characterized by their specific cellular and biological content (in particular, a high density of macrophages), rather than by their impact on the vessel lumen. Early identification of high-risk plaques may be useful for preventing ischemic events. One major hurdle in detecting high-risk atherosclerotic plaques in coronary arteries is the lack of an imaging modality that allows for the identification of atherosclerotic plaque composition with high spatial and temporal resolutions. Here we show that macrophages in atherosclerotic plaques of rabbits can be detected with a clinical X-ray computed tomography (CT) scanner after the intravenous injection of a contrast agent formed of iodinated nanoparticles dispersed with surfactant. This contrast agent may become an important adjunct to the clinical evaluation of coronary arteries with CT.     

Arteriosclerosis in the influx and intravisceral arteries of the liver, kidney and lung of WHHL rabbits.

We performed a histopathological investigation on arteriosclerotic development in the influx and intravisceral arteries of the liver, kidney and lung of male WHHL rabbits. In the influx arteries of these organs, we observed severe atherosclerotic vascular lesions with high-grade luminal stenosis. In the intravisceral arteries of the liver and kidney, no arteriosclerotic lesions were observed. However, in the intrapulmonary arteries, we recognized severe atherosclerotic vascular changes with high-grade stenosis or total obstruction of the lumen in some middle to large sized pulmonary arteries. These observations indicate that the development of arteriosclerosis in parenchymatous organs differs, and that some organs are predisposed to arteriosclerosis formation.     

Neovascularization of coronary tunica intima (DIT) is the cause of coronary atherosclerosis. Lipoproteins invade coronary intima via neovascularization from adventitial vasa vasorum, but not from the arterial lumen: a hypothesis

ABSTRACT: BACKGROUND: An accepted hypothesis states that coronary atherosclerosis (CA) is initiated by endothelial dysfunction due to inflammation and high levels of LDL-C, followed by deposition of lipids and macrophages from the luminal blood into the arterial intima, resulting in plaque formation. The success of statins in preventing CA promised much for extended protection and effective therapeutics. However, stalled progress in pharmaceutical treatment gives a good reason to review logical properties of the hypothesis underlining our efforts, and to reconsider whether our perception of CA is consistent with facts about the normal and diseased coronary artery. Analysis To begin with, it must be noted that the normal coronary intima is not a single-layer endothelium covering a thin acellular compartment, as claimed in most publications, but always appears as a multi-layer cellular compartment, or diffuse intimal thickening (DIT), in which cells are arranged in many layers. If low density lipoprotein cholesterol (LDL-C) invades the DIT from the coronary lumen, the initial depositions ought to be most proximal to blood, i.e. in the inner DIT. The facts show that the opposite is true, and lipids are initially deposited in the outer DIT. This contradiction is resolved by observing that the normal DIT is always avascular, receiving nutrients by diffusion from the lumen, whereas in CA the outer DIT is always neovascularized from adventitial vasa vasorum. The proteoglycan biglycan, confined to the outer DIT in both normal and diseased coronary arteries, has high binding capacity for LDL-C. However, the normal DIT is avascular and biglycan-LDL-C interactions are prevented by diffusion distance and LDL-C size (20 nm), whereas in CA, biglycan in the outer DIT can extract lipoproteins by direct contact with the blood. These facts lead to the single simplest explanation of all observations: (1) lipid deposition is initially localized in the outer DIT; (2) CA often develops at high blood LDL-C levels; (3) apparent CA can develop at lowered blood LDL-C levels. This mechanism is not unique to the coronary artery: for instance, the normally avascular cornea accumulates lipoproteins after neovascularization, resulting in lipid keratopathy. Hypothesis Neovascularization of the normally avascular coronary DIT by permeable vasculature from the adventitial vasa vasorum is the cause of LDL deposition and CA. DIT enlargement, seen in early CA and aging, causes hypoxia of the outer DIT and induces neovascularization. According to this alternative proposal, coronary atherosclerosis is not related to inflammation and can occur in individuals with normal circulating levels of LDL, consistent with research findings.     

Development of atherosclerotic lesions in cholesterol-loaded rabbits.

To examine both of the target vessels and the optimal time of their endothelial denudation to study vascular restenosis after balloon injury in cholesterol-loaded rabbits, we made 36 atherosclerotic rabbits by feeding a hypercholesterol diet, and histologically examined the onset time and the development of atherosclerosis. Atheromatous changes were observed first after the 5th week in the thoracic aorta from the start of the diet, and then extended to the abdominal aorta, coronary artery with time. The atherosclerotic lesions in the thoracic aorta and the proximal portion of the coronary artery showed high-grade concentric intimal thickening with luminal stenosis. The abdominal aortic lesion mildly progressed. In the renal, carotid and femoral arteries, in contrast, slight atheroscleromatous changes developed during the diet period. These results suggest that the thoracic and abdominal aortas and the coronary artery would be suitable as target vessels to study vascular restenosis after balloon injury, and the endothelial denudation of these vessels should be performed between the 8th and 15th week in this diet protocol for an accurate analysis.     

A proteomic focus on the alterations occurring at the human atherosclerotic coronary intima

Coronary atherosclerosis still represents the major cause of mortality in western societies. Initiation of atherosclerosis occurs within the intima, where major histological and molecular changes are produced during pathogenesis. So far, proteomic analysis of the atherome plaque has been mainly tackled by the analysis of the entire tissue, which may be a challenging approach because of the great complexity of this sample in terms of layers and cell type composition. Based on this, we aimed to study the intimal proteome from the human atherosclerotic coronary artery. For this purpose, we analyzed the intimal layer from human atherosclerotic coronaries, which were isolated by laser microdissection, and compared with those from preatherosclerotic coronary and radial arteries, using a two-dimensional Differential-In-Gel-Electrophoresis (DIGE) approach. Results have pointed out 13 proteins to be altered (seven up-regulated and six down-regulated), which are implicated in the migrative capacity of vascular smooth muscle cells, extracellular matrix composition, coagulation, apoptosis, heat shock response, and intraplaque hemorrhage deposition. Among these, three proteins (annexin 4, myosin regulatory light 2, smooth muscle isoform, and ferritin light chain) constitute novel atherosclerotic coronary intima proteins, because they were not previously identified at this human coronary layer. For this reason, these novel proteins were validated by immunohistochemistry, together with hemoglobin and vimentin, in an independent cohort of arteries.     

The number of cells and the cell proliferation in intima of various human arteries

Increased cell proliferation at the early stages of an atherosclerotic lesion is considered an important stage of development of this pathology, but the degree of the proliferation at various stages of formation of atherosclerotic plaque in various human large arteries so far has been studied insufficiently. In the present work, we studied the thickness of intima and proliferation of the newly “infiltrated” hematogenic and resident cells in atherosclerotic lesion of carotid and coronary arteries; a comparison is also made with similar results obtained on the aorta and presented in our earlier publications. Analysis of thickness of intima and of proliferation in normal intima and at various stages of atherosclerotic lesion (initial stages, lipid strips, lipofibrous plaques, fibrous plaques) showed that, in spite of similar tendencies toward changing the level of infiltration of hematogenic cells and proliferation in various types of arteries, there exist significant quantitative differences between various types of arteries. Thus, it is found that hematogenic cells in lipofibrous plaques of coronary and carotid arteries account for one-third and almost half of the total cell population, respectively, whereas the atherosclerosis-lesioned sites of the aorta, as we showed earlier, contain no more than 15% of hematogenic cells. This allows one to think that the contribution of hemopoietic cells to development of atherosclerosis in carotid and coronary arteries is greater than in the aorta. In spite of differences in the number of the hemopoietic cells accumulating in intima, an analogous bell-shaped dependence of the number of cells on the type of lesion (in the sequence normal intima-initial stages of pathology-lipid strips-lipofibrous plaques-fibrous plaques) was shown for coronary and carotid arteries. Visualization of proliferating (PCNA-positive) cells in atherosclerosed and normal (unchanged) zones of coronary and carotid arteries revealed a similar picture. The maximum number of PCNA-positive resident cells was found in lipofibrous plaques. Changes of the total number of cells were accompanied by a change in the number of proliferating resident and proliferating hematogenic cells.     

Long-term estradiol treatment improves VIP-mediated vasodilation in atherosclerotic proximal coronary arteries

The aim of the present study was to evaluate the impact of long-term estrogen replacement therapy (ERT) on the vasodilatory effect of the two peptides vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) in atherosclerotic coronary and cerebral arteries.Female ovariectomized homozygous Watanabe heritable hyperlipidemic rabbits were randomized to 16 weeks treatment with 17beta-estradiol or placebo. The diet was semisynthetic, thereby avoiding the influence of phytoestrogens. Artery ring segments were mounted for isometric tension recordings in myographs. Following precontraction, the dose-response relationships for VIP and PACAP were evaluated.Treatment with 17beta-estradiol significantly improved the maximum VIP-mediated vasodilation (E(max), percentage of precontraction) in proximal coronary arteries (45.8+/-9.6% vs. 24.1+/-3.7%, p<0.05). In the same artery segment, 17beta-estradiol induced a significant decrease in the relative ratio between the repeated contractile response to potassium 30 and 120 mM (100+/-7% vs. 132+/-11%, p<0.05). For distal coronary arteries, there was a tendency to similar changes, but no statistical differences for the potassium or VIP responses in cerebral or distal coronary arteries were found between the two groups. 17beta-estradiol induced no changes in the PACAP-mediated vasodilation.These results suggest that long-term treatment with 17beta-estradiol improves the VIP-mediated but not the PACAP-mediated vasodilation in atherosclerotic proximal coronary arteries.     

Damage and regeneration of the endothelium of the aorta in experimental hypercholesterolemia

In dynamics of the experimental hypercholesterolemia in rabbits, peculiarities of endothelial regeneration have been studied. Comparison of proliferative activity level in endotheliocytes with structural-functional state of the endothelial monolayer at atherogenesis makes it possible to consider, that the lesion of the endothelium cannot be regarded as an initiating factor for formation of atherosclerotic lesions. Formation of the lesions in the internal lining of the arteries is preceded by certain disorders in permeability of the endothelial barrier at increasing concentration of cholesterin in blood plasma, accompanying with a sharp activation of the cell proliferative activity. When lipid plates and atherosclerotic plaques are already formed, the processes of the endothelial damage and regeneration occur in parallel. The regeneration is ensured with an intensive proliferation and growth of endotheliocytes onto deendotheliolized areas of the damaged intima.     

Numbers of cells and cell proliferation in intima of different human arteries

Increased cell proliferation in early atherosclerotic lesions is recognized as an essential event of atherogenesis but the levels of cell proliferation in different stages of atherosclerotic plague formation in different types of human large arteries are still insufficiently studied. In the present work, we studied intima thickness and proliferation of newly "infiltrates" hematogenous and resident cells in atherosclerotic lesions of the carotid and coronary arteries and compared these parameters with those in the aorta, reported by us in earlier publication. Analysis of intima thickness and proliferation in grossly unaffected intima and in different types pf atherosclerotic lesions (initial lesions, fatty streaks, lipofibrous, plaques, and fibrous plaque) revealed that although there were similar tendencies in the change of the infiltration levels of hematogenous cells and proliferation in different types of arteries, there were significant quantitative differences between different types of arteries. Hematogenous cells in lipofibrous plaques of the coronary and carotid arteries were found to account for a third and almost for a half of the total cell population, respectively, while atherosclerotic lesions in the aorta, as it has been shown by us earlier, to contain no more than 15% ofhematogenous cells. This suggests that the contribution of hematogenous cells to the development of atherosclerosis in the carotid and the coronary artery appears to be more significant than that in the aorta. Despite the differences in numbers of accumulating hematogenous cells in the intima, a similar "bell-shaped" dependence of cell numbers on the lesion type, involved in the following sequence: unaffected intima-initial lesions-fatty streaks-lipofibrous plaques-fibrous plaques, was detected in the coronary and carotid arteries. The visualization of proliferating cells (PCNA-positive) in atherosclerotic and unaffected zones of the coronary and carotid arteries revealed similar patterns. The maximum numbers of PCNA-positive resident cells were identified in lipofibrous plaques. The changes in the total cell numbers were accompanied by the changes in the numbers of both proliferating resident cells and proliferating hematogenous cells.     

A new model of chronic cardiac ischemia in rabbits.

Chronic cardiac ischemia has mainly been studied in large species such as pigs or dogs. Little research has been performed using small species such as rabbits. In the present study, 1-3 wk after implantation of a novel device (ameroid) on the circumflex coronary artery of New Zealand White rabbits, vessel patency was evaluated by coronary angiography, corrosion cast, and radiolabeled microspheres. Coronary angiograms showed, after 21 days, either total occlusion or severe stenosis in seven of eight arteries, which was confirmed by corrosion casts. The ameroid group had less blood flow in the epicardial (-62%) and endocardial (-54%) layers of the ischemic area compared with sham-operated rabbits (P < 0.05). Blood flow increased in the ischemic area compared with day 0 during acute occlusion, suggesting that progressive coronary occlusion initiated the growth of de novo collateral vessels. Thus we have developed a new model of chronic cardiac ischemia in rabbits with documented progressive coronary stenosis and occlusion that is suitable to test various therapeutic angiogenesis strategies.     

Antibody against cholesteryl ester transfer protein (CETP) elicited by a recombinant chimeric enzyme vaccine attenuated atherosclerosis in a rabbit model

The recombinant chimeric enzyme of AnsB-TTP-CETPC, which comprised asparagianse, tetanus toxin helper T-cell epitope and CETP B-cell epitope, was used to vaccinate New Zealand white rabbits in alum adjuvant. After anti-CETP antibodies were successfully produced, rabbits were fed with a high cholesterol diet for fifteen weeks until atherosclerotic lesions formed in arteries. The results showed that after CETP was inhibited by anti-CETP antibodies the fraction of plasma cholesterol in HDL increased and the fraction of plasma cholesterol in LDL decreased in rAnsB-TTP-CETPC immunized rabbits. The average size of aorta atherosclerotic plaques in rabbits treated with rAnsB-TTP-CETPC was 42.3% less than in rabbits treated with OVA (neutral control), or 47.6% less than in rabbits treated with rHSP 65 (negative control). The average thickness of hyperplastic coronary artery in rAnsB-TTP-CETPC immunized rabbits was 159+/-12 microm, which was significantly lower than in rHSP 65 immunized rabbits (187+/-15 microm) or OVA immunized rabbits (248+/-18 microm). The data reported here demonstrated that rAnsB-TTP-CETPC could significantly attenuate the development of atherosclerosis in rabbits fed with high cholesterol diet, and might be developed to an anti-atherosclerosis vaccine in the future.     

Proteomics analysis of human coronary atherosclerotic plaque: a feasibility study of direct tissue proteomics by liquid chromatography and tandem mass spectrometry

Cardiovascular disease presents significant variations in human populations with respect to the atherosclerotic plaque progression, inflammation, thrombosis, and rupture. To gain a more comprehensive picture of the pathogenic mechanism of atherosclerosis and the variations seen in patients, efficient methods to identify proteins from the normal and diseased arteries need to be developed. To accomplish this goal, we tested the feasibility and efficiency of protein identification by a recently developed method, termed direct tissue proteomics (DTP). We analyzed frozen and paraformaldehyde-fixed archival coronary arteries with the DTP method. We also validated the distinct expression of four proteins by immunohistochemistry. In addition, we demonstrated the compatibility of the DTP method with laser capture microdissection and the possibility of monitoring specific cytokines and growth factors by the absolute quantification of abundance method. Major findings from this feasibility study are that 1) DTP can be used to efficiently identify proteins from paraformaldehyde-fixed, paraffin-embedded, and frozen coronary arteries; 2) approximately twice the number of proteins were identified from the frozen sections when compared with the paraformaldehyde-fixed sections; 3) laser capture microdissection is compatible with DTP; and 4) detection of low abundance cytokines and growth factors in the coronary arteries required selective reaction monitoring experiments coupled to absolute quantification of abundance. The analysis of 35 human coronary atherosclerotic samples allowed identification of a total of 806 proteins. The present study provides the first large scale proteomics map of human coronary atherosclerotic plaques.     

兔颈动、静脉移植桥血管狭窄模型的建立及其电镜观察

目的:建立兔颈动、静脉移植血管桥动物模型,观察移植桥血管内膜增生和狭窄的电镜下表现。方法:通过兔双侧颈动脉进行动脉桥和静脉桥的移植,形成双侧移植血管桥再狭窄动物模型。在第8周施行血管桥移植手术的同时留取右侧颈动静脉标本作为对照血管,再分别于第12周、16周和第20周分别处死模型兔,采集移植桥血管标本,在光镜下测量其内膜厚度、面积、狭窄度,并进行电镜观察。结果:颈动脉和颈静脉桥移植后,随着时间的延长,桥血管的出现平滑肌迁移,脂质沉积,内膜增生,血管狭窄等改变,且以静脉桥血管的病理改变更为明显。结论:在兔形成动脉粥样硬化病变基础上,进行双侧颈动脉血管桥的移植,建立兔双侧颈动脉移植血管桥再狭窄动物模型,有利于设立自身对照,研究术后动静脉桥再狭窄差异机制;建立动、静脉桥后,位于血管中膜的平滑肌细胞出现向血管内膜迁移现象,说明中膜平滑肌细胞迁移进入内膜导致新内膜形成是血管再狭窄的重要环节。     

共聚焦激光扫描显微镜活体观察动脉粥样硬化家兔模型大脑皮质血管内血液缘流厚度的改变

目的:探讨在兔脑皮层动脉粥样硬化时血管内缘流厚度的变化。方法:用共聚焦激光扫描显微镜比较家兔脑皮质动脉内缘流厚度在正常、粥样硬化病理状态下的不同。结果:在血管直径为74.87±3.26μm的血管中血液缘流厚度在病理状态下随血管的舒张、收缩而发生改变;而在血管直径为94.33±2.84μm的缘流厚度在血管收缩时发生改变。结论:动脉粥样硬化的家兔模型中血液缘流厚度随血液的流变性质改变而改变。     

液氮冻伤术建立动脉粥样硬化破裂斑块及血栓模型

目的创建一种操作简单、经济实用的动脉粥样硬化（AS）破裂斑块及血栓动物模型。方法21只雄性纯种新西兰白兔随机分为两组：液氮冻伤＋高脂喂养组（A组=11只）和高脂喂养组（B组=10只）。A组实施右颈总动脉内膜液氮冻伤术结合高脂饲料喂养,B组单纯给予高脂饲料喂养。8周末以液氮激发斑块破裂,激发前后分别采血检测血脂、hsC-RP、MMP-9及PAI-1水平;激发48h后处死所有动物,取出右颈总动脉作HE染色及免疫组化染色等,光镜及电镜观察破裂斑块及血栓形成情况。结果8周后兔血脂水平明显升高;激发后血浆hsC-RP、MMP-9及PAI-1均明显升高;所有A组兔子的右颈总动脉均可见AS破裂斑块及血栓形成,而B组兔子未见斑块或血栓形成;所建立的破裂斑块在组织结构、细胞构成、生长特征和脂质沉积方面与人类斑块相似。结论液氮冻伤术能简便、快速、高效地建立AS破裂斑块及血栓模型,从而为研究人类AS破裂斑块及血栓形成的机理和药物干预治疗提供了一种新型动物模型。     

Everolimus-induced mTOR inhibition selectively depletes macrophages in atherosclerotic plaques by autophagy

Current pharmacological approaches to stabilize nonobstructive rupture-prone atherosclerotic plaques have only partially reduced the incidence of acute coronary syndromes and sudden death. Macrophages in these vulnerable plaques play a pivotal role in plaque destabilization, whereas smooth muscle cells promote plaque stability. In a recent study, we report that implantation of stents eluting everolimus, a mammalian target of rapamycin (mTOR) inhibitor, in atherosclerotic arteries of cholesterol-fed rabbits, led to a marked reduction in macrophage content without altering the amount of smooth muscle cells. Our in vitro studies showed that treatment of macrophages and smooth muscle cells with everolimus induced inhibition of translation of both cell types. However, cell death occurred only in macrophages and was characterized by bulk degradation of long-lived proteins, processing of microtubule associated protein light chain 3 (LC3), and cytoplasmic vacuolization, which are all markers of autophagy. Everolimus-induced autophagy was mediated by mTOR inhibition because cell viability was not affected using tacrolimus, an mTOR independent everolimus-analogue. These results provide proof-of-principle that macrophages in the vascular wall can be selectively cleared via induction of autophagy by mTOR inhibition. Therefore, stent-based delivery of an mTOR inhibitor may be a promising novel strategy for treatment of vulnerable atherosclerotic plaques.     

Preliminary observations, at the ultrastructural level, on the reactivity of cerebral arteries from New Zealand rabbits to combined atherogenic stimuli (hypercholesterol diet and hypertension)

Both in monkeys (Rhesus and Cynomolgus) and in New Zealand rabbits fed an atherogenic diet, a marked delay in the appearance of atherosclerotic lesions of the cerebral arteries in comparison with other arterial districts has been observed. This appearance has been described in monkeys as relatively earlier if hypertension is added to the atherogenic diet. Preliminary observations on a little group of rabbits on a 3 months hypercholesterolic diet, subjected to Goldblatt aortic coarctation, have shown an increase of blood pressure and a severe gross atherosclerotic involvement of aorta, resembling the one obtainable after 6 months of atherogenic diet. Histologically, the aorta predominantly shows lesions of the fatty streaks type with necrotic areas in the deep; the carotid lesions show some lipid in smooth muscle cells disseminated in a sub-endothelial "edematous" space (rich in protein). The cerebral arteries do not show any lesion. At TEM, the aortic lesions look sometimes as advanced plaques with an initial fibrosis at the surface; the carotid lesions are characterized by a granular deposit in the sub-endothelial space in which some smooth muscle cells (with lipid in the cytoplasm) are present; in the cerebral arteries only the presence of collagen fibers among the smooth muscle cells of the media, never observed in the animals fed the atherogenic diet alone, has sometimes been noted.     

C3 deposition in cholesterol-induced atherosclerosis in rabbits: a possible etiologic role for complement in atherogenesis.

Hypercholesterolemia was induced in rabbits by feeding Purina Chow supplemented with cholesterol (5 g/kg body weight/day). The serum cholesterol levels of these rabbits increased progressively and after 3 to 5 months were 4 to 9-fold greater than those of the control animals. Decrease in total hemolytic complement was not apparent during the feeding regimen. Morphologic examination of aortae of these hypercholesterolemic rabbits showed typical atherosclerotic intimal plaques. Immunofluorescent microscopy with fluorescein (F)-labeled anti-rabbit C3 showed deposition of C3 in the intimal and inner medial layers as early as 3 months on high cholesterol diet. C3 deposits were also observed in the renal glomeruli and in the walls of coronary arteries. However, fluorescent studies failed to demonstrate the presence of IgG, IgM, and C4 at these sites. Tissues from control animals fed normal diets were negative for immunoglobulins, C3, and C4. These results suggest that the complement system may be implicated in the pathogenesis of cholesterol-induced atherosclerosis in rabbits.     

Natriuretic peptide system gene expression in human coronary arteries.

The natriuretic peptides (NPs) ANF, BNP, and CNP have potent anti-proliferative and anti-migratory effects on vascular smooth muscle cells (SMCs). These properties make NPs relevant to the study of human coronary atherosclerosis because vascular cell proliferation and migration are central to the pathophysiology of atherosclerosis. However, the existence and cytological distribution of NPs and their receptors in human coronary arteries remain undetermined. This has hampered the development of hypotheses regarding the possible role of NPs in human coronary disease. We determined the pattern of expression of NPs and their receptors (NPRs) in human coronary arteries with atherosclerotic lesions classified by standard histopathological criteria as fatty streak/early atherosclerotic lesions, intermediate plaques, or advanced lesions. The investigation was carried out using a combination of immunocytochemistry (ICC), in situ hybridization (ISH), and semi-quantitative polymerase chain reaction (PCR). Both by ICC and ISH, ANF was found in the intimal and medial layers of all lesions. BNP was highly expressed in advanced lesions where it was particularly evident by a strong ISH signal but weak ICC staining. CNP was demonstrable in all types of lesions, giving a strong signal by ISH and ICC. This peptide was particularly demonstrable in the endothelium, as well as in the SMCs of the intima, media, and vasa vasorum of the adventitia and in macrophages. By ISH, NPR-A was not detectable in any of the lesions but both NPR-B and NPR-C were found in the intimal and the inner medial layers. By RT-PCR, mRNA levels of all NPs tended to be increased in macroscopically diseased arteries, but only the values for BNP were significantly so. No significant changes in NPR mRNA levels were detected by PCR. In general, the signal intensity given by the NPs and their receptors by ICC or ISH appeared dependent on the type of lesion, being strongest in intermediate plaques and decreasing with increasing severity of the lesion. This study constitutes the first demonstration of NPs and NPR mRNAs in human coronary arteries and supports the existence of an autocrine/paracrine NP system that is actively modulated during the progression of atherosclerotic coronary disease. This suggests that the coronary NP system is involved in the pathobiology of intimal plaque formation in humans and may be involved in vascular remodeling.