Expression of leptin receptor (Ob-R) in human atherosclerotic lesions: potential role in intimal neovascularization

Neovascularization of the adventitial vasa vasorum with extension into the intima of atherosclerotic lesions is frequently observed, but its pathophysiological significance is still subject to debate. Recently, leptin, the product of the Ob gene, was identified. Leptin, via activation of the endothelial receptor (Ob-R), generates a growth signal involving a tyrosine kinase-dependent intracellular pathway and promotes angiogenic processes. We hypothesized that a high concentration of leptin within vasa vasorum and plaque itself, may influence inflammatory and vascular neovascularization coupling with functional upregulation of the vascular endothelial growth factor (VEGF). Microscopic computerized tomography was utilized for the spatial distribution of vasa vasorum and intimal neovascularization from atherosclerotic human coronary arteries. Atherosclerotic coronary arteries showed a dense plexus of microvessels in the adventitia and plaque itself. Microscopic analysis from human atherosclerotic aortas revealed an increase in the intimal thickness with neovascularization. The immunoreactivity for Ob-R, VEGF and matrix metalloproteinase (MMP) increased in atherosclerotic plaque, predominantly in the endothelial lining of the intimal neovessel and macrophages/foam cells. Our observation of a prominent colocalization between Ob-R, VEGF and MMP supports this hypothesis and these factors participate in the neovascularization of atherosclerotic lesions. The present study is the first report on vascular tissue and it opens a promising perspective concerning future investigations of leptin-dependent modulation of atherogenesis and vascular neovascularization under pathophysiolgical conditions.     

冠状动脉外膜炎症在动脉粥样硬化病灶形成中的作用

目的：探讨动脉外膜炎症诱发载脂蛋白E基因剔除（apoE-/-）小鼠冠状动脉粥样硬化病灶发生的机制。 方法： 取apoE-/-小鼠心脏做连续切片，选择冠状动脉外膜有炎性细胞浸润的3类部位代表粥样硬化病灶形成过程中的3个阶段：①未发现粥样硬化病灶；②有直接从主动脉延伸的病灶顶端和③成熟粥样硬化病灶的冠状动脉。分别采用HE染色、Movat染色、免疫组化和透射电镜方法鉴定3个病变阶段的冠状动脉外膜中炎细胞类型。 结果： ①未发现粥样硬化病灶；②有直接从主动脉延伸的病灶顶端和③成熟粥样硬化病灶的冠状动脉外膜分别以巨噬细胞、中性粒细胞和淋巴细胞浸润为主，其构成比分别为60.00%、57.65%和66.67%，各组构成比分别与其它两组比较均有显著差异（P<0.01）。 结论： 动脉外膜炎症可能是诱发动脉粥样硬化发生的早期事件之一，ApoE-/-小鼠冠状动脉粥样硬化病灶形成过程中，动脉外膜经历了一个从急性炎症到慢性炎症的过程。     

载脂蛋白E基因敲除小鼠动脉粥样硬化早期血管外膜成纤维细胞增殖活性增强

目的:探讨动脉外膜成纤维细胞增殖与早期动脉粥样硬化病灶形成的关系。方法:选择6周龄载脂蛋白E基因敲除[apoE(-/-)]小鼠和野生型C57BL/6小鼠,高脂喂养2、4和10周后,在各个时点处死动物前24 h经腹腔注射5-溴-2-脱氧尿嘧啶(BrdU),后选取升主动脉制备连续切片,通过HE染色观察组织形态学的变化,用免疫组化方法观察不同时点血管外膜及内膜BrdU的表达变化。体外培养高脂喂养2周的apoE(-/-)小鼠和C57BL/6小鼠动脉外膜成纤维细胞,通过BrdU掺入法测定细胞增殖活性,流式细胞术测定细胞周期。结果:体内实验发现apoE(-/-)小鼠高脂喂养2周后,在无可见内膜病灶形成之前,首先在主动脉外膜发现BrdU标记的阳性细胞,之后才在损伤内膜观察到BrdU标记细胞。而C57BL/6小鼠在任何时点都未检测到BrdU标记的细胞。体外实验观察到apoE(-/-)小鼠血管外膜成纤维细胞BrdU标记的细胞数显著多于C57BL/6小鼠(P0.01),apoE(-/-)小鼠血管外膜成纤维细胞S期及G2/M期所占百分比明显高于对照组(P0.05)。结论:血管外膜成纤维细胞增殖可能参与早期动脉粥样硬化病灶形成。     

Development of intimal lesions after leukocyte migration into the vascular wall.

A model was developed to study the role of leukocytes in the development of vascular lesions. Implantation of an endotoxin-soaked cotton thread in the adventitia on the ventral side of the rat femoral artery resulted in leukocyte migration into the vessel wall exclusively in the ventral half of the vessel. Leukocyte migration occurred from both the luminal and adventitial side and consisted of neutrophils and mononuclear cells. Smooth muscle cell rich intimal lesions localized to the ventral half of the vessel were first observed 1 week after implantation. Lesions remained localized to the ventral half of the vessel wall through the 6th week. When leukocyte migration into the vessel wall was inhibited by treatment with dexamethasone, lesion development did not occur. These results suggest that leukocytes can stimulate smooth muscle cell migration into the intima and result in intimal lesion formation.     

A model of primary atherosclerosis and post-angioplasty restenosis in mice

Although mice deficient in various genes are providing greater insight into the mechanisms of restenosis after angioplasty, there have been limitations with murine models not simulating human vascular disease. To develop a more clinically applicable model of primary atherosclerosis and restenosis following angioplasty of the primary lesion, we fed apolipoprotein E-deficient mice a Western diet and occluded the left common carotid artery for 2 days. Three weeks after flow was restored, the temporarily occluded carotids demonstrated atherosclerotic lesions containing foam cells, cholesterol clefts, necrotic cores, and fibrous capsules. The atherosclerotic carotids in other animals underwent angioplasty with a beaded probe, resulting in plaque and medial layer disruption. Three weeks after angioplasty, although there was significant neointimal lesion formation, the luminal narrowing did not change significantly secondary to overall vessel enlargement (positive remodeling). Neointimal lesions were composed of smooth-muscle cells and extracellular matrix observed adjacent to the original atherosclerotic plaques. Similarly, even at 3 months after the angioplasty the lumen was maintained despite greater neointimal lesion formation caused by progressive positive remodeling. This new murine model of primary atherosclerosis and postangioplasty intimal hyperplasia and remodeling mimics the human disease pattern of postangioplasty intimal hyperplasia. Used in transgenic animals, this model will likely facilitate understanding of the mechanisms of restenosis in humans.     

An immunohistochemical analysis of fibroblasts in giant cell arteritis

BackgroundGiant cell arteritis (GCA) is a systemic vasculitis of large and medium vessels characterized by an inflammatory arterial infiltrate. GCA begins in the adventitia and leads to vascular remodeling by promoting proliferation of myofibroblasts in the intima. The morphology of the fibroblasts in the adventitia in GCA is unclear. Access to temporal artery biopsies allows morphological studies and evaluation of the microenvironment of the arterial wall. We evaluated the distribution of vascular fibroblasts and of markers of their activation in GCA.MethodsFormalin-fixed paraffin-embedded tissue sections from 29 patients with GCA and 36 controls were examined. Immunohistochemistry was performed for CD90, vimentin, desmin, alpha-smooth muscle actin (ASMA), prolyl-4-hydroxylase (P4H), and myosin to evaluate the distribution of fibroblasts within the intima, media, and adventitia.ResultsTemporal arteries from patients with GCA showed increased levels of CD90, vimentin, and ASMA in the adventitia and intima compared to the controls. Desmin was expressed only in the media in both groups. P4H was expressed similarly in the adventitia and intima in the two groups. Adventitial and intimal CD90⁺ cells co-expressed P4H, ASMA, and myosin at a high level in GCA.ConclusionThe results suggest a role for adventitial fibroblasts in GCA. Inhibiting the differentiation of adventitial fibroblasts to myofibroblasts has therapeutic potential for GCA.     

TWO AUTOPSY CASES OF "PULSELESS DISEASE"

Two cases of "Pulseless Disease" are reported. The main lesions In this condition are localized to the arteries of elastic type including pulmonary artery. The chief changes in the initial stage are limited to the adventitia and outer portion of media, based on an inflammatory reaction, which in turn brings about several following changes in arterial wall; marked fibrous thickening of adventitia, destruction of media and intimal thickening. In "Pulseless Disease," the lumen of the affected artery tends to become stenosed or obstructed, but can also be dilated and partially aneurysm-like. The stenosis of the lumen is chiefly caused by the contraction of the adventitial fibrosis overcoming the pressure on the arterial wall, promoted with the intimal thickening. Dilatation of that may be caused by increased arterial pressure, which exceedes the contracting effect of the wall, and is also influenced by the duration of the disease.     

Ultrastructural localization of tissue factor on monocyte-derived macrophages and macrophage foam cells associated with atherosclerotic lesions

The expression of tissue factor (TF) antigen by circulating monocytes, cultured macrophages, and macrophages associated with atherosclerotic lesions was ultrastructurally analysed using immunogold labeling. A subpopulation of macrophages associated with the intimal surface overlying lesions had a significant TF expression. Macrophages and macrophage foam cells that projected from the intima into the arterial lumen also expressed a high level of TF (14-fold increase over control). In contrast, circulating monocytes and macrophages in culture did not express TF above background control levels. This TF expression by macrophages in vivo but not by macrophages cultured from either normal or hypercholesterolemic animals suggests that monocyte activation and macrophage transition, as measured by TF expression, is lesion-dependent and not stimulated solely by intimal attachment, surface migration, or hypercholesterolemia. These results further suggest that macrophages and foam cells associated with early lesions of atherosclerosis can initiate fibrin formation, which could contribute to lesion complications and transition to a fibromuscular stage.     

Adventitial inflammation: a possible pathogenic link to the instability of atherosclerotic plaque

A variety of cells, including fibroblasts, mast cells, macrophages, and ganglionic cells, are present in coronary artery adventitia. In the infarct-related coronary arteries of myocardial infarction patients, the majority of mast cells are found in the outer layer of the adventitia. Neurogenic stimulation of mast cells in the adventitia of coronary arteries may release vasoactive compounds, such as histamine and leukotrienes, which can contribute to the complex neurohormonal response that leads to abnormal coronary vasoconstriction. Lymphocytes and bacteria are also present mainly in the adventitial layer. Chlamydia pneumoniae is directly involved in the development of adventitial and plaque inflammation (pan-arteritis), leading to plaque rupture. Adventitial O(2)(-) may also play an extensive role in the control of vascular tone. Therefore, adventitial inflammation may play a pivotal role for atherosclerotic lesion development and atheroma instability.     

Development of complex atherosclerotic lesions in pigs with inherited hyper-LDL cholesterolemia bearing mutant alleles for apolipoprotein B.

The development of atherosclerotic lesions was studied in pigs aged 4 to 54 months with inherited hyperlow-density lipoprotein (LDL) and hypercholesterolemia (IHLC pigs). These pigs bear the Lpb5 and Lpu1 mutant alleles for apolipoproteins B and U and demonstrate spontaneously elevated cholesterol levels, due primarily to elevated LDL. By 1 year of age, IHLC pigs exhibited focal lesions in the major coronary, iliac, and femoral arteries that were composed of macrophage-derived from cells and smooth muscle cells. Peripheral arterial lesions were more fibrous than those found in the coronaries. By 2 years of age, complicated stenotic lesions containing fibrous caps, necrotic cores, cholesterol clefts, granular calcium deposits, and neovascularization deep within the lesion were common in the major coronary vessels. Peripheral vascular lesions were more smooth muscle cell-rich and fibrotic. By 3 years of age, neovascularization was observed throughout the intimal lesion, and hemorrhage and rupture were common. The extent of complicated lesion formation correlated with both the degree and duration of hypercholesterolemia, with the most stenotic lesions observed in the coronary arteries of the oldest animals having the highest cholesterol levels. Thus IHLC pigs with mutant apolipoproteins B and U develop complicated atherosclerotic plaques that closely resemble advanced atherosclerotic lesions found in humans.     

Immunohistochemical study of intimal microvessels in coronary atherosclerosis.

Two hundred ninety-nine human coronary artery paraffin-embedded tissue blocks were examined for intimal microvessel invasion by probing for factor VIII-associated antigen with indirect immunofluorescence and high resolution confocal microscopy. The results obtained confirm that intimal microvessels originate in the adventitia and show that the richness of intimal microvessels is strongly positively correlated with intimal thickness and negatively correlated with relative lumen size. A number of plasma constituents were examined in serial sections. Comparison of immunofluorescence distribution patterns of these components with intimal microvessel distribution patterns reveals that intimal microvessels leak plasma albumin into artery walls, exude fibrinogen, and are associated with the build-up of plasma cells within atherosclerotic lesions. Therefore, intimal microvessels are demonstrated to play important roles in the development of atherosclerosis.     

Long-term engraftment of bone marrow-derived cells in the intimal hyperplasia lesion of autologous vein grafts

Intimal hyperplasia of autologous vein grafts is a critical problem affecting the long-term patency of many types of vascular reconstruction. Within intimal hyperplasia lesions, smooth muscle cells are a major component, playing an essential role in the pathological process. Given that bone marrow-derived cells may differentiate into smooth muscle cells in the neointima of injured arteries, we hypothesized that the bone marrow may serve as a source for some of the smooth muscle cells within intimal hyperplasia lesions of vein grafts. To test this hypothesis, we used an established mouse model for intimal hyperplasia in wild-type mice that had been transplanted with bone marrow from a green fluorescent protein (GFP+/+) transgenic mouse. High-resolution confocal microscopy analysis performed 2 and 8 weeks after grafting demonstrated expression of GFP in 5.4 +/- 0.8% and 11.9 +/- 2.3%, respectively, of smooth muscle cells within intimal hyperplasia lesions. By 16 weeks, GFP expression in smooth muscle cells was not detected by immunohistochemistry; however, real-time PCR revealed that 20.2 +/- 1.7% of the smooth muscle cells captured from the neointima lesion by laser capture microdissection at 16 weeks contained GFP DNA. Our results suggest that bone marrow-derived cells differentiated into smooth muscle cells within the intimal lesion and may provide a novel clinical approach for decreasing intimal hyperplasia in vein grafts.     

Risk factors for atherosclerosis and the development of preatherosclerotic intimal hyperplasia

BACKGROUND: Intimal hyperplasia or thickening is considered to be the precursor lesion for atherosclerosis in humans; however, the factors governing its formation are unclear. To gain insight into the etiology of preatherosclerotic intimal hyperplasia, we correlated traditional risk factors for atherosclerosis with the intimal hyperplasia in an atherosclerosis-resistant vessel, the internal thoracic artery. METHODS: Paired internal thoracic arteries were obtained from 89 autopsies. Multivariate logistic regression and multiple regression models were used to examine the association of preatherosclerotic intimal hyperplasia with traditional risk factors for atherosclerosis: age, gender, hypertension, smoking, body mass index, diabetes, and hypercholesterolemia. RESULTS: Atherosclerotic lesions consisting of fatty streaks and/or type III intermediate lesions were identified in 19 autopsies. Only age >75 years was found to be significantly correlated with atherosclerotic lesion development (P=.01). Multiple regression model of the intima/media ratio in all 89 cases revealed age >75 years (P<.0001), age 51-75 years (P=.0012), smoking (P=.008), and hypertension (P=.02) to be significantly correlated with intimal thickness. In the 70 cases without atherosclerosis, only age 51-75 years (P=.006) and smoking (P=.028) were found to be significantly associated with preatherosclerotic intimal thickening. CONCLUSIONS: In the atherosclerosis-resistant internal thoracic artery, preatherosclerotic intimal hyperplasia routinely forms during adulthood after the fourth decade and is associated with at least two traditional risk factors for atherosclerosis: age and smoking. These observations indicate that in some settings, intimal hyperplasia may be part of the disease process of atherosclerosis and that its formation may be influenced by traditional risk factors for atherosclerosis.     

Histologic findings in specimens obtained by percutaneous directional coronary atherectomy.

Percutaneous directional coronary atherectomy is a second-generation, catheter-based technique for the treatment of coronary artery disease that provides a unique opportunity to study tissue obtained from coronary artery stenoses. We reviewed the histologic findings in 131 coronary atherectomy specimens from 116 patients, including 79 primary lesions and 52 restenosis lesions that developed after a prior coronary intervention. Although atherosclerotic plaque was seen in 95% of cases, an important observation was the finding of deep arterial wall components in the majority of patients, including media in 61% and adventitia in 31%. Despite the relatively common finding of deep wall components, this was not associated with acute clinical complications. Intimal hyperplasia was seen in 64% of cases, including 96% of restenosis lesions. However, intimal hyperplasia was also seen in 45% of primary lesions. The intimal hyperplasia in primary and restenosis lesions was histologically identical and was characterized by cells that showed staining for vimentin and muscle-specific actin, consistent with a myogenous and/or myofibroblastic phenotype. Restenosis was seen in 29 of 94 lesions (31%) with angiographic follow-up an average of 6.3 months after atherectomy. The risk of restenosis was not significantly related to the vessel treated, the nature of the lesion treated (ie, primary v restenosis lesion), or any of the histologic features evaluated. In conclusion, (1) deep arterial wall components are commonly observed in coronary atherectomy specimens without acute complications and (2) intimal hyperplasia is a sensitive, but not specific, marker for restenosis lesions.     

Angiogenesis and lymphangiogenesis and expression of lymphangiogenic factors in the atherosclerotic intima of human coronary arteries

Little information regarding the development of lymphangiogenesis in coronary atherosclerosis is available. We immunohistochemically investigated the correlation among intimal neovascularization (CD34 for angiogenesis and lymphatic vessel endothelial hyaluronan receptor-1 [LYVE-1] and podoplanin for lymphangiogenesis), the expression of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGF-D), and the progression of atherosclerosis using 169 sections of human coronary arteries from 23 autopsy cases. The more the atherosclerosis advanced, the more often the neointimas contained newly formed blood vessels ( P < .0001). Vascular endothelial growth factor-C was expressed mostly in foamy macrophages and in some smooth muscle cells, whereas VEGF-D was abundantly expressed in both. The number of VEGF-C-expressing cells, but not that of VEGF-D-expressing cells, was increased as the lesion advanced and the number of intimal blood vessels increased ( P < .01). Lymphatic vessels were rare in the atherosclerotic intima (LYVE-1 vs CD34 = 13 vs 3955 vessels) compared with the number seen in the adventitia (LYVE-1 vs CD34 = 360 vs 6921 vessels). The current study suggests that VEGF-C, but not VEGF-D, may contribute to plaque progression and be a regulator for angiogenesis rather than lymphangiogenesis in coronary atherosclerotic intimas. Imbalance of angiogenesis and lymphangiogenesis may be a factor contributing to sustained inflammatory reaction during human coronary atherogenesis.     

Learning from rejection: What transplantation teaches us about (other) vascular pathologies

Allograft vasculopathy is an accelerated intimal hyperplastic lesion leading to progressive vascular stenosis; it represents the major long-term limitation to successful solid organ transplant. Although allograft vasculopathy is not formally an autoimmune disease, nor does it constitute a major cause of cardiovascular disease on a purely numerical basis, its pathogenesis provides an important window on the mechanisms by which immune injury can drive more common vascular pathologic entities. Thus, insights gleaned from vascularized solid organ transplants can shed new mechanistic (and therapeutic) light on: 1) the intimal vascular responses accompanying typical atherosclerosis and other inflammatory vessel diseases (e.g., scleroderma); 2) the pathogenesis of vascular stenosis versus aneurysm formation; 3) the sources of intimal smooth muscle cells in the healing of any vascular injury; and 4) the mechanisms by which smooth muscle cells are recruited into intimal lesions. Indeed, research on allograft vasculopathy has led to the understanding that interferon-γ plays a similar pathogenic role in a host of vascular stenosing lesions—and that Th2 cytokines can drive vascular remodeling and aneurysm formation. Moreover, circulating precursors (and not just medial smooth muscle cells) contribute to the intimal hyperplasia seen in atherosclerosis and in-stent restenosis. That non-vessel smooth muscle cells can be recruited to sites of vessel injury further suggests that chemokine and adhesion molecule interactions may be viable targets to limit vascular stenosis in a wide range of vascular lesions. This review will describe the pathogenesis of allograft vasculopathy, and will relate how understanding the underlying pathways informs our understanding of both human transplant-associated disease, as well as other human vascular pathologies.     

The role of the monocyte in atherogenesis: I. Transition of blood-borne monocytes into foam cells in fatty lesions.

In a previous publication the author and his co-workers demonstrated that atherosclerotic lesion development in the aorta of hypercholesterolemic pigs was preceded by intimal penetration of blood-borne mononuclear cells, and that medial smooth muscle cells were not involved in the formation of early fatty lesions in this model. The current study shows that aortic arch lesions do not progress beyond the fatty cell lesion stage for up to 30 weeks of a moderate cholesterol/lard diet, although they become more extensive in area. Mononuclear cells were found adherent to the endothelium, in endothelial junctions, and in the intima during this period, and were ultrastructurally identified as monocytes by the presence of peroxidase-positive granules (peroxisomes) in their cytoplasm. In addition, lesion areas with nonspecific esterase activity correlated well with Sudan IV staining. Intimal monocytes and altered intimal monocytes with an enlarged cytoplasm and containing a few lipid droplets were both shown to be phagocytic by their uptake of ferritin, which had penetrated the intima after intravenous injection. Circulating monocytes and those adherent to the endothelial surface did not contain ferritin in these animals. The results indicate that blood mononuclear cells associated with lesion formation in this model are, in fact, monocytes, which subsequently undergo transformation into macrophage foam cells in fatty streak lesions. The absence of medial cell involvement indicates that monocytes are the major foam cell precursor in these lesions.     

血管树突状细胞在人主动脉粥样硬化早期病变中的分布

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

A hypothesis: adiponectin mediates anti-atherosclerosis via adventitia-AMPK-iNOS pathway

Adiponectin is an adipocyte-derived protein with insulin-sensitizing, anti-inflammatory, and anti-atherogenic properties and is abundantly found in plasma. Vascular adventitia is the outermost connective and supporting tissue of vessels. Recently, increasing evidence has shown that infection in the adventitia is one of the causes of atherosclerosis and restenosis. Our previous study indicated that local transferring adenovirus expressing adiponectin gene (Ad-APN) to intima and adventitia can suppress atherosclerosis, but the exact mechanism is still obscure. We speculate that with infection in the adventitia, adiponectin can activate AMP-activated protein kinase (AMPK) through adiponectin receptors in the membranes of adventitial fibroblasts and then inhibit the expression and activity of inducible nitric oxide synthase (iNOS); secretion of adventitial infective factors; division, proliferation and translation of adventitial fibroblasts; and change of adventitial fibroblasts to myofibroblasts, finally decreasing oxidative/nitrative stress to reduce atherosclerotic plaque area and stabilize atherosclerotic plaques. The proposition may provide clues into the development of a novel treatment for atherosclerosis.     

Composition and classification of human atherosclerotic lesions

Summary Human atherosclerotic disease can be resolved into eight types of lesion, each characterized by its composition and structure and the absence or degree of intimal injury. The eight types have been arranged in the sequence in which they may progress in complexity from the initial change in childhood or youth to the clinical endpoints in older persons. While lesions at first increase primarily by intra- and extracellular accumulation of lipid, this in itself rarely accounts for symptomatic obstruction. Lipidic lesions become symptomatic primarily by means of successively superimposed deposits of thrombotic material. Non-homogeneity of hemodynamic forces within the length of an artery account for local differences in intima thickness (adaptive intimal thickening) and, in persons with risk factors, differences in susceptibility to lesion formation. According to the degree to which they can accumulate or retain lipid and bring about secondary mechanisms, specific locations of the arterial tree have been designated asatherosclerosisresistant, atherosclerosis-prone and progression-prone.