The human carotid atherosclerotic plaque stimulates angiogenesis on the chick chorioallantoic membrane.

The chick chorioallantoic membrane was used to determine whether the carotid atherosclerotic plaque stimulates angiogenesis. Carotid endarterectomy specimens (1 mm3) with fibromuscular plaque (n = 8) and complicated plaque (n = 11) were implanted on the membrane on day nine of incubation and the response evaluated on day 11. Following fixation in situ with 10% formalin the angiogenic response was evaluated by: (1) examining whole membrane mounts, (2) quantitatively from a vascular density index and (3) from a histological study. Unmanipulated chorioallantoic membrane (n = 11) and plaque boiled prior to implantation (n = 6) served as controls. The vascularity of whole mounts of both fibromuscular and complicated plaque was greater than the controls. Vessel density of the membrane was estimated by counting the number of vessels intersecting four concentric circles (144.5 mm total circumference) placed on the formalin fixed membrane. The vascular density index due to the fibromuscular plaque (390.6 +/- 8.3) and complicated plaque (391.0 +/- 14.9) were similar (P greater than 0.9) but were significantly greater (P less than 0.001) than the unmanipulated membrane (327.9 +/- 5.6) or after treatment with the boiled plaque (283.8 +/- 15.6). Transforming growth factor beta 1 confirmed the validity of the experimental model to study angiogenesis. The histology of the chorioallantoic membrane due to either type of plaque was similar. Numerous vessels surrounded the plaque, and intraplaque vessels containing nucleated chick erythrocytes were observed. Although scattered vessels surrounded the boiled plaque, intraplaque vessels were not observed. This study demonstrates that the atherosclerotic plaque has angiogenic properties that may account for the increase in vasa vasorum that is associated with the plaque.     

Atherosclerotic plaque progression and vulnerability to rupture: angiogenesis as a source of intraplaque hemorrhage

Observational studies of necrotic core progression identify intraplaque hemorrhage as a critical factor in atherosclerotic plaque growth and destabilization. The rapid accumulation of erythrocyte membranes causes an abrupt change in plaque substrate characterized by increased free cholesterol within the lipid core and excessive macrophage infiltration. Neoangiogenesis is associated closely with plaque progression, and microvascular incompetence is a likely source of intraplaque hemorrhage. Intimal neovascularization is predominantly thought to arise from the adventitia, where there are a plethora of pre-existing vasa vasorum. In lesions that have early necrotic cores, the majority of vessels invading from the adventitia occur at specific sites of medial wall disruption. A breech in the medial wall likely facilitates the rapid in-growth of microvessels from the adventitia, and exposure to an atherosclerotic environment stimulates abnormal vascular development characterized by disorganized branching and immature endothelial tubes with "leaky" imperfect linings. This network of immature blood vessels is a viable source of intraplaque hemorrhage providing erythrocyte-derived phospholipids and free cholesterol. The rapid change in plaque substrate caused by the excessive accumulation of erythrocytes may promote the transition from a stable to an unstable lesion. This review discusses the potential role of intraplaque vasa vasorum in lesion instability as it relates to plaque rupture.     

颈动脉粥样硬化斑块内新生血管的研究现状及进展

颈动脉粥样硬化斑块破裂是导致脑卒中的重要原因之一,大量研究证实颈动脉斑块内新生血管是导致斑块内出血、斑块破裂的重要因素。炎症因子及各类细胞通过斑块内新生血管进入斑块,导致斑块稳定性破坏,但影响斑块内新生血管形成的重要相关因子和主要机制目前尚未完全明确,因此识别斑块内新生血管、探索斑块内新生血管形成的相关因子及机制是研究斑块内新生血管致斑块不稳定性的关键。抑制斑块内新生血管生成,可能成为防治颈动脉斑块破裂、降低脑栓塞事件发生的新策略。本综述旨在探讨颈动脉斑块内新生血管形成的相关因子、机制以及检测成像的最新研究进展,为动脉粥样硬化的诊疗提供支持。     

Atherosclerotic plaque rupture: local or systemic process?

It is generally established that the unstable plaque is the major cause of acute clinical sequelae of atherosclerosis. Unfortunately, terms indicating lesions prone to plaque instability, such as "vulnerable plaque," and the different phenotypes of unstable plaques, such as plaque rupture, plaque fissuring, intraplaque hemorrhage, and erosion, are often used interchangeably. Moreover, the different phenotypes of the unstable plaque are mostly referred to as plaque rupture. In the first part of this review, we will focus on the definition of true plaque rupture and the definitions of other phenotypes of plaque instability, especially on intraplaque hemorrhage, and discuss the phenotypes of available animal models of plaque instability. The second part of this review will address the pathogenesis of plaque rupture from a local and a systemic perspective. Plaque rupture is thought to occur because of changes in the plaque itself or systemic changes in the patient. Interestingly, contributing factors seem to overlap to a great extent and might even be interrelated. Finally, we will propose an integrative view on the pathogenesis of plaque rupture.     

Platelets enter atherosclerotic plaque via intraplaque microvascular leakage and intraplaque hemorrhage: a histopathological study in carotid plaques

BackgroundPlatelets foster an inflammatory environment that influences atherosclerotic lesion progression and facilitates plaque rupture, in addition to their role in acute thrombus formation. The route of entry of platelets into the atherosclerotic plaque and their exact location inside the plaque are however not completely understood.Methods and results188 carotid plaques were examined for the presence of platelets using immunohistochemistry (CD42b), and 76/188 (40.4%) were platelet positive. Platelets were observed in intraplaque hemorrhages, around plaque microvessels, mostly without leakage of erythrocytes; and in mural thrombi. Platelet positive staining was associated with a higher plaque microvessel density, and elevated plaque-levels of interleukin-8.ConclusionDue to their short life span, platelets reflect recent bleeding. It can be hypothesized that platelets might serve as a marker for leaky microvessels inside atherosclerotic plaques that are at risk for development, or progression of plaque hemorrhage.     

Type 1 diabetes promotes disruption of advanced atherosclerotic lesions in LDL receptor-deficient mice

Cardiovascular disease, largely because of disruption of atherosclerotic lesions, accounts for the majority of deaths in people with type 1 diabetes. Recent mouse models have provided insights into the accelerated atherosclerotic lesion initiation in diabetes, but it is unknown whether diabetes directly worsens more clinically relevant advanced lesions. We therefore used an LDL receptor-deficient mouse model, in which type 1 diabetes can be induced at will, to investigate the effects of diabetes on preexisting lesions. Advanced lesions were induced by feeding mice a high-fat diet for 16 weeks before induction of diabetes. Diabetes, independently of lesion size, increased intraplaque hemorrhage and plaque disruption in the brachiocephalic artery of mice fed low-fat or high-fat diets for an additional 14 weeks. Hyperglycemia was not sufficient to induce plaque disruption. Furthermore, diabetes resulted in increased accumulation of monocytic cells positive for S100A9, a proinflammatory biomarker for cardiovascular events, and for a macrophage marker protein, without increasing lesion macrophage content. S100A9 immunoreactivity correlated with intraplaque hemorrhage. Aggressive lowering primarily of triglyceride-rich lipoproteins prevented both plaque disruption and the increased S100A9 in diabetic atherosclerotic lesions. Conversely, oleate promoted macrophage differentiation into an S100A9-positive population in vitro, thereby mimicking the effects of diabetes. Thus, diabetes increases plaque disruption, independently of effects on plaque initiation, through a mechanism that requires triglyceride-rich lipoproteins and is associated with an increased accumulation of S100A9-positive monocytic cells. These findings indicate an important link between diabetes, plaque disruption, and the innate immune system.     

Elimination of neoangiogenesis for plaque stabilization: is there a role for local drug therapy?

Emerging data suggest that intraplaque hemorrhage is critical in promoting atherosclerotic lesion instability. Because red blood cell membranes are a rich source of free cholesterol and accumulated red blood cells within plaques promote inflammation, intraplaque hemorrhage is associated with expansion of the necrotic core. Plaque hemorrhage results from the development of immature neointimal vasa vasorum. Therefore, it is proposed that molecular therapies designed to eliminate pathologic neovascularization within developing lesions will interrupt the process of hemorrhage and decrease the rate of necrotic core expansion. The elimination of intraplaque neovascularization would involve targeting of pre-existing and new vessel development. The concept of vascular regression has met some success in other neovascular-dependent diseases, including macular degeneration and malignancies. The efficacy of this novel approach is dependent on gaining critical knowledge of the environment required to support development and maturation of the vasa vasorum within varying plaque types. A multitargeted approach involving selective local antiangiogenic agents should contribute to prevention of plaque progression and its clinical consequences.     

Neovascular Endothelial-Cell Activation: The Link Between Angiogenesis,Intimal Leukocyte Content,and Development of Symptomatic Carotid Occlusive Disease

New blood vessels in atherosclerotic lesions are postulated to be responsible for plaque instability by acting as conduits for inflammatory cells. This study assessed the association between macrophage content of plaque caps of carotid atherosclerotic lesions, plaque vascularity, and endothelial-cell activation within these blood vessels. Carotid endarterectomy specimens from patients who underwent endarterectomy for carotid occlusive disease (ten symptomatic, six asymptomatic) were examined. Sequential transverse sections were obtained and were stained conventionally and immunohistochemically with CD 34 (an endothelial-cell marker) and CD 68 (a macrophage marker), as well as VCAM-1 and ICAM-1 (markers of endothelial-cell activation). Microvessel and macrophage counts were performed for the whole plaque and the plaque cap respectively. The differences in microvessel density, macrophage content, and ICAM-1 and VCAM-1 expression, between symptomatic and asymptomatic patients and in different plaque types were examined, as was the association between microvessel density and microvessel content of atherosclerotic lesions. Higher microvessel counts were observed in patients with symptomatic carotid occlusive disease (p < 0.01). Higher ICAM-1 and VCAM-1 expression was noted in new blood vessels in plaques obtained from patients with symptomatic carotid occlusive disease, compared with asymptomatic patients (p < 0.001 and p < 0.01 respectively). A close association was observed between macrophage content of the plaque cap and microvessel counts in atherosclerotic lesions (p < 0.0001), as well as ICAM-1 (p < 0.001) and VCAM-1 (p < 0.001) expression, in these microvessels. This study highlights the significance of neovascularity and endothelial-cell activation in the evolution of symptomatic carotid occlusive disease. Furthermore, it points to a relationship between the inflammatory process and angiogenesis within the plaque, which may be mediated through endothelial-cell activation.     

Pathology of the thin-cap fibroatheroma: a type of vulnerable plaque

Thin cap atheroma is the precursor of plaque rupture, which accounts for a majority of coronary thrombi. The morphologic features of thin cap atheromas that predict rupture are unknown, but we know from studies of ruptured plaques that large necrotic cores, fibrous cap < 65 microns and numerous macrophages within the cap likely indicate instability. There is some evidence that a speckled pattern of calcification is associated with vulnerability to rupture. There are usually multiple thin cap atheroma in the hearts of patients dying with acute plaque rupture, as well as multiple fibroatheromas with intraplaque hemorrhage. Targeted therapy for the purpose of stabilizing coronary lesions that are prone to rupture is a major future goal of the interventionist.     

Histopathological characterization of carotid plaques echotomography

The positive correlation between cerebral ischemia and carotid atherosclerosis of extracranial tract has been well established. The reliability of echotomography as diagnostic and prognostic tool in the evaluation of the carotid atherosclerotic lesions is now intensively investigated. Most of the attention has been payed to the accuracy in the determination of the carotid stenosis. A percentage of cerebrovascular accidents do not correlate to the vascular stenosis but to the other modifications induced by the atherosclerotic plaque, such as the release of emboli, acute stenosis for intraplaque haemorrhage or thrombosis. Thus the evaluation of the composition of the plaque may represent a good prognostic tool. In the present study, fifty-two carotid obtained at surgery from patients, who preoperatively underwent ultrasonographic scanning of both carotid arteries, were examined by histological methods. In calcified tissues a significant correlation between findings obtained with both techniques was observed. In the complicated plaques atheromasic gruel, intraplaque haemorrhages and thrombosis could not be discriminated by echotomography.     

Melatonin stabilizes rupture‐prone vulnerable plaques via regulating macrophage polarization in a nuclear circadian receptor RORα‐dependent manner

Rupture of vulnerable plaques is the main trigger of acute cardio‐cerebral vascular events, but mechanisms responsible for transforming a stable atherosclerotic into a vulnerable plaque remain largely unknown. Melatonin, an indoleamine hormone secreted by the pineal gland, plays pleiotropic roles in the cardiovascular system; however, the effect of melatonin on vulnerable plaque rupture and its underlying mechanisms remains unknown. Here, we generated a rupture‐prone vulnerable carotid plaque model induced by endogenous renovascular hypertension combined with low shear stress in hypercholesterolemic ApoE^?/? mice. Melatonin (10 mg/kg/d by oral administration for 9 weeks) significantly prevented vulnerable plaque rupture, with lower incidence of intraplaque hemorrhage (42.9% vs. 9.5%, P = 0.014) and of spontaneous plaque rupture with intraluminal thrombus formation (38.1% vs. 9.5%, P = 0.029). Mechanistic studies indicated that melatonin ameliorated intraplaque inflammation by suppressing the differentiation of intraplaque macrophages toward the proinflammatory M1 phenotype, and circadian nuclear receptor retinoid acid receptor‐related orphan receptor‐α (RORα) mediated melatonin‐exerted vasoprotection against vulnerable plaque instability and intraplaque macrophage polarization. Further analysis in human monocyte‐derived macrophages confirmed the role of melatonin in regulating macrophage polarization by regulating the AMPKα‐STATs pathway in a RORα‐dependent manner. In summary, our data provided the first evidence that melatonin‐RORα axis acts as a novel endogenous protective signaling pathway in the vasculature, regulates intraplaque inflammation, and stabilizes rupture‐prone vulnerable plaques.     

Imaging of the vasa vasorum

Neovascularization of the arterial walls by adventitial vasa vasorum appears to participate in the process of atherosclerosis progression and destabilization. Although the biological mechanisms associated with plaque instability are still unclear, the uncontrolled formation of intraplaque neovessels appears to contribute to the development of complex atheromatous lesions. Recent reports have described the use of several ultrasound-based techniques for the real-time detection of intraplaque neovascularization. Preliminary studies in animal models have shown that the detection and characterization of adventitial neovascularization are technically feasible. The further development of these imaging techniques relies on the successful implementation of contrast microspheres capable of enhancing microvascular structures. These contrast agents serve as surrogate red blood cells and perform acoustically as true intravascular tracers providing, in real-time, the amount and distribution of neovessels within atherosclerotic lesions. Several ultrasound-based techniques are under development for the detection of adventitial vasa vasorum in the carotid and coronary vascular territories. Although still in early validation phases, these techniques might permit the early diagnosis and stratification of subclinical atherosclerosis, thus permitting aggressive preventive therapy. In the near future, innovative contrast agents using specific ligands are likely to expand the diagnostic and therapeutic possibilities of these emerging imaging techniques.     

Intraplaque MMP-8 levels are increased in asymptomatic patients with carotid plaque progression on ultrasound

Carotid atherosclerotic plaque remodelling and increased risk of symptomatic plaque rupture seem to be partially mediated by matrix metalloproteinases (MMPs). In this study, we have investigated whether different MMPs are related to carotid atherosclerosis or to recent ischaemic brain disease. Eighty-four consecutive patients undergoing carotid endarterectomy for symptomatic and asymptomatic disease were studied. Plaques were analysed by ultrasound and later by morphology. Plasma MMP-2, MMP-8 and MMP-9 levels were quantified by ELISA. MMP expression and activity in carotid plaques was analysed by Western blotting and in situ zymography. Results were analysed with respect to plaque stability, morphology, symptomatic disease, presence of vascular risk factors and plasma markers of acute inflammation as high sensitivity C-reactive protein (hsCRP), fibrinogen, D-dimer and white blood cell counts. Patients with hypoechogenic plaques on ultrasound had more plasma MMP-8 (p = 0.04) and increased MMP activity as assessed by in situ zymography. Asymptomatic patients with plaque progression had more active intraplaque MMP-8 than asymptomatic patients without plaque progression. Presence of recent intraplaque haemorrhage or past history of CAD was related to increased activity of MMPs as assessed by in situ zymography (p < 0.01, CI 95% 0.8-1.0). Plasma MMP-8 and MMP-9, but not MMP-2 levels, decrease with time after ischaemic stroke. Patients with hypertension had more intraplaque active MMP-9 than normotensive (p = 0.03, CI 95% 0.7-1.0). Hypoechogenic carotid plaques had increased MMP activity and asymptomatic patients with plaque progression show increase intraplaque MMP-8 levels.     

Pathology of human plaque vulnerability: Mechanisms and consequences of intraplaque haemorrhages

Atherothrombotic diseases are still major causes of inability and mortality and fighting atherothrombosis remains a public health priority. The involvement of repeated intraplaque haemorrhages (IPH) in the evolution of atherothrombotic lesions towards complications was proposed as early as 1936. This important topic has been recently revisited and reviewed. Histological observations have been corroborated by magnetic resonance imaging (MRI) of human carotid atheroma, identifying IPH as the main determinant of plaque evolution towards rupture.     

超声造影评价颈动脉斑块内新生血管的研究进展

动脉粥样硬化斑块与心脑血管事件的发生密切相关,早期评估动脉粥样硬化斑块的稳定性对于预防心脑血管事件发生及制定治疗方案十分重要。大量研究证实斑块内新生血管与斑块的稳定性有关,超声造影可显示和评估颈动脉斑块内新生血管,是近年来斑块稳定性研究的热点技术之一。     

炎症反应在易损斑块中的作用及其机制研究进展

炎症反应在易损斑块的形成和进展中发挥重要作用,同时调控血管局部病变及全身炎症状态。一些促炎性细胞和炎症因子使斑块纤维帽的抗张强度降低,坏死脂质内核增大,血管机械稳定性丧失和斑块破裂;另一方面,炎症反应的激活和代谢紊乱也会引起内皮功能不全、斑块侵蚀进而导致血栓形成。该过程主要由巨噬细胞和淋巴细胞等多种炎症细胞参与,并受到多种因素调控,包括胆固醇结晶和脂质递质、血管剪切力、血管新生及斑块内出血等。此外,机体还存在一些抑炎性分子,能避免易损斑块向破裂或侵蚀进展。促炎和抗炎反应的平衡影响急性冠状动脉事件的发生。因此,以炎症反应为靶点,筛选出有易损斑块的患者并干预,或可减少急性冠状动脉事件的发生和改善预后,具有重要临床价值。     

Thioredoxin in coronary culprit lesions: possible relationship to oxidative stress and intraplaque hemorrhage

The present study investigated the expression of thioredoxin (TRX), an important anti-oxidative protein, and its relationship to plaque instability in atherectomy specimens from 43 and 42 patients with stable (SAP) and unstable (UAP) angina pectoris, respectively. We histologically assessed thrombus formation, cellular elements, localization of TRX and of oxidized low density lipoprotein (ox-LDL), intraplaque hemorrhage, and transition metal iron (Fe(2+), Fe(3+)) deposition in these specimens. The clinical characteristics of the two groups did not differ except for aspirin administration. The incidence of thrombus formation was more frequent (P=0.005) and immunopositive areas of macrophage, TRX and ox-LDL were significantly larger in patients with UAP than SAP (P<0.001, each). Macrophages were mainly immunoreactive for TRX and ox-LDL. Intraplaque hemorrhage evaluated by glycophorin A immunoreactivity and Fe(2+)/Fe(3+) deposition was also more obvious in lesions from patients with UAP than SAP (P<0.001, each). Additionally, immunopositive areas of TRX and ox-LDL positively correlated with Fe(2+)/Fe(3+) deposition and were also associated with thrombus formation. Although the underlying mechanisms remain unknown, TRX was up-regulated in response to increased oxidative stress and associated with intraplaque hemorrhage of coronary culprit lesions, and thus might be a potent marker of plaque instability.     

Antiangiogenic therapy for normalization of atherosclerotic plaque vasculature: a potential strategy for plaque stabilization

Angiogenesis within human atherosclerotic plaques has an important role in plaque progression as immature blood vessels leak red blood cells and inflammatory mediators into the plaque center. Accumulation of free cholesterol from red blood cell membranes potentially increases the size of the necrotic core and triggers a chain of events that promote plaque destabilization. Antiangiogenic agents have been shown to prune some tumor vessels and 'normalize' the structure and function of the remaining vasculature, thereby improving the access of chemotherapeutic agents to tumors. We propose that antiangiogenic therapy can similarly stabilize vulnerable 'rupture-prone' plaques by pruning and normalizing immature intraplaque vessels, preventing further intraplaque hemorrhage. This normalization would limit necrotic core enlargement, further luminal narrowing and the degree of inflammation. Such normalization has been realized using vascular endothelial growth factor antagonists for the treatment of cancer and age-related macular degeneration. The development of this novel approach to prevent plaque progression might add to the armamentarium of preventive measures for acute myocardial infarction, stroke and sudden cardiac death.     

Echodensitometry: a methodologic approach to the non-invasive diagnosis of carotid atherosclerotic plaques

The ultrasound image of the fibroatheromatous plaque has to date been unable to reveal the presence or absence of complications as shown by histology, such as thrombosis and intraplaque hemorrhage. We propose a novel densitometric method for determining the composition of the plaque at the echotomography, along with a new classification based on mathematical models drawn from the optical density distribution curve. This approach avails of an extremely high sensitivity. Percent areas occupied by thrombosis, intraplaque hemorrhage and atheroma in histologic sections have been shown to correspond to hyporeflecting areas in echotomographic images, whereas those occupied by calcium and fibrous tissue correspond to reflecting areas with or without acoustic shadow, respectively. This method may prove to have an important role in the non-invasive monitoring of even slight changes during progression or regression of the fibroatheromatous plaque.     

Initiation of angiogenesis in atherosclerosis: smooth muscle cells as mediators of the angiogenic response to atheroma formation

Neovascularization of atherosclerotic lesions favors their progression toward rupture. Despite this pathophysiological importance, data regarding the mechanism(s) initiating plaque neovascularization are scarce. Recent findings indicate that smooth muscle cells located underneath early aortic atheromatous lesions display a pro-angiogenic phenotype, and that lipid mediators derived from these lesions are potent inducers of this phenotypic change. Here, we discuss these new data suggesting that smooth muscle cells could be the central organizers of an angiogenic response initiated by the very first cause of the atheromatous disease, the accumulation and retention of lipids in the arterial wall.