Matrix vesicles in the fibrous cap of atherosclerotic plaque: possible contribution to plaque rupture.

Plaque rupture is the most common type of plaque complication and leads to acute ischaemic events such as myocardial infarction and stroke. Calcification has been suggested as a possible indicator of plaque instability. Although the role of matrix vesicles in the initial stages of arterial calcification has been recognized, no studies have yet been carried out to examine a possible role of matrix vesicles in plaque destabilization. Tissue specimens selected for the present study represented carotid specimens obtained from patients undergoing carotid endarterectomy. Serial frozen cross-sections of the tissue specimens were cut and mounted on glass slides. The thickness of the fibrous cap (FCT) in each advanced atherosclerotic lesion, containing a well developed lipid/necrotic core, was measured at its narrowest sites in sets of serial sections. According to established criteria, atherosclerotic plaque specimens were histologically subdivided into two groups: vulnerable plaques with thin fibrous caps (FCT <100 microm) and presumably stable plaques, in which fibrous caps were thicker than 100 microm. Twenty-four carotid plaques (12 vulnerable and 12 presumably stable plaques) were collected for the present analysis of matrix vesicles in fibrous caps. In order to provide a sufficient number of representative areas from each plaque, laser capture microdissection (LCM) was carried out. The quantification of matrix vesicles in ultrathin sections of vulnerable and stable plaques revealed that the numbers of matrix vesicles were significantly higher in fibrous caps of vulnerable plaques than those in stable plaques (8.908+0.544 versus 6.208+0.467 matrix vesicles per 1.92 microm2 standard area; P= 0.0002). Electron microscopy combined with X-ray elemental microanalysis showed that some matrix vesicles in atherosclerotic plaques were undergoing calcification and were characterized by a high content of calcium and phosphorus. The percentage of calcified matrix vesicles/microcalcifications was significantly higher in fibrous caps in vulnerable plaques compared with that in stable plaques (6.705+/-0.436 versus 5.322+/-0494; P= 0.0474). The findings reinforce a view that the texture of the extracellular matrix in the thinning fibrous cap of atherosclerotic plaque is altered and this might contribute to plaque destabilization.

Cellular destruction following transmyocardial laser revascularization (TMR).

During transmyocardial revascularization, cellular destruction of cardiomyocytes occurs as a result of the high-energy laser. However, the features of myocardial cellular destruction are unclear. The present study was undertaken to examine the structural characteristics of cell death in the myocardium following transmyocardial revascularization. Myocardial specimens from 3 male patients who had died within 11 days following laser revascularization were collected within 1 h of death and were analyzed by immunohistochemistry and electron microscopy. For immunohistochemistry, antibodies to pro-apoptotic proteins CPP32 and BAX were used. Immunohistochemical examination demonstrated the presence of cells expressing both CPP32 and BAX along the laser channel. Electron microscopic analysis revealed that the lining surface of laser channels consisted of condensed acellular debris and dead cells. No endothelialization of channels was noted. The lumen of laser channels were surrounded by a rim of acellular debris with several outer concentric rims of cardiomyocytes showing features of cellular destruction. The present study identified features of both necrotic and apoptotic cellular death following laser revascularization.

Hallmarks of atherosclerotic lesion development with special reference to immune inflammatory mechanisms.

Atherosclerosis is a complex disease that starts during childhood and becomes clinically evident later in life. Atherosclerosis results from an interaction between diverse factors including lipid metabolism, blood coagulation elements, cytokines and hemodynamic stress. Humoral and cellular immune reactions are key events in pathogenesis with the balance between pro-inflammatory and anti-inflammatory stimuli being crucial for the development of atherosclerotic lesions. Atherosclerotic lesions are characterised by the presence of immune competent cells including T-cells and dendritic cells. Dendritic cell interactions with T-cells might be responsible for T-cell activation in atherogenesis. Despite increasing acknowledgment that immune-inflammatory processes participate in atherogenesis, the immune mechanisms remain incompletely defined.

Identification of dendritic cells in aortic atherosclerotic lesions in rats with diet-induced hypercholesterolaemia.

We have previously identified dendritic cells (DCs) in the intima of human large arteries. These vascular DCs are common in atherosclerotic lesions but their immature forms are also present in normal arterial intima. Pathophysiological studies on vascular DCs are limited because they have only been studied in human specimens obtained at operation or post-mortem. The aim of the current study was to determine whether DCs participate in the development of atherosclerotic lesions in hypercholesterolemic rats. Male Wistar rats were divided into a control (n=13) and experimental cohort (n=48). The experimental animals were fed an atherogenic diet and 1% saline, while the controls were fed standard rat cubes and water. The aortas were obtained from both groups at 10, 20, and 30 weeks following commencement of the diet. An en face immunohistochemical technique, routine section immunohistochemistry, and transmission electron microscopy were used to detect the presence of DCs in the aortas. Examination of the aortas showed that S100+ cells with dendritic cell morphology were present in the aortic intima of hypercholesterolemic rats. The S100+ DCs displayed immunopositivity for OX-62 and MHC Class II antibodies. Within various types of atherosclerotic lesions, these cells were clustered throughout the intima but were especially prominent around arterial branch-points where they co-localized with various cell types, including T-cells and macrophages. Ultrastructural analysis confirmed the presence of cells with characteristics typical of DCs. These features included the presence of a well-developed tubulovesicular system, dendritic processes, and a lack of secondary lysosomes and phagosomes. This study establishes the presence of DCs in the aortic intima of rats with diet-induced atherosclerosis. The presence of DCs in this model of experimental atherogenesis could provide a new approach to investigating the function of DCs and may help clarify the immune-inflammatory mechanisms underlying atherosclerosis.

A comparative thrombogenicity study of heparin soaked fluoro-passivated polyester and ePTFE patches in sheep.

OBJECTIVES: to compare heparin soaked fluoro-passivated gelatine sealed polyester and expanded polytetrafluoroethylene (ePTFE) patches in a sheep model of acute platelet accumulation following patch angioplasty. MATERIALS AND METHODS: heparin soaked patches were placed in the carotid arteries of 9 sheep and autologous (111)Indium labelled platelets were infused. The patches were explanted two hours after the injection of labelled platelets. Median specimen radioactivity was calculated as a ratio of radioactivity in explanted and in 4 ml of blood. Explanted patches were also investigated by scanning electron microscopy (SEM). RESULTS: platelet accumulation was significantly greater on ePTFE patches. For both materials platelet accumulation was greater at the distal end compared to the proximal (p<0.05). SEM demonstrated more platelets as well as thicker thrombus layer on ePTFE-patches. CONCLUSION: in sheep carotid arteries, a fluoropassivated gelatine sealed polyester patch appears to result in less platelet accumulation when compared to ePTFE.

Ernst Ferdinand Sauerbruch: rise and fall of the pioneer of thoracic surgery.

Ferdinand Sauerbruch (1875-1951) was a pioneer of thoracic and cardiac surgery and is undoubtedly one of the twentieth century's most outstanding surgeons. Before 1904 operations on the thorax met with fatal complications due to pneumothorax. Sauerbruch developed a pressure-differential chamber that maintained normal respiration and enabled safe operations to be undertaken on the thorax. Together with von Mikulicz, he initiated intrathoracic operations and later developed various surgical procedures on the mediastinum, lungs, pericardium, heart, and esophagus. The simple yet effective techniques of positive-pressure ventilation replaced the expensive, cumbersome negative-pressure chamber. Sauerbruch's latter years were marred by dementia that adversely affected his personality, intellect, and capacity as a surgeon. The unjustifiable toll of increasing patient morbidity and mortality forced authorities to dismiss him in 1949. He died at the age of 76 in Berlin. After almost a century since the advent of the first safe thoracic surgery, the advances in technique and technology have been enormous. A great deal is owed to the inspiration and contributions of Ferdinand Sauerbruch.

Expression of vascular endothelial growth factor in aortocoronary saphenous vein bypass grafts.

Neovascularisation is a prominent feature of long-term aortocoronary saphenous vein bypass grafts but mechanisms involved in the formation of neovessels have not been previously studied. Vascular Endothelial Growth Factor (VEGF) is an important angiogenic factor that induces migration and proliferation of endothelial cells, enhances permeability and modulates thrombogenecity. This study investigated the expression of VEGF in aortocoronary saphenous vein bypass grafts. Aortocoronary saphenous vein bypass grafts with angiographic luminal stenosis of >75% were explanted from 14 patients at redo coronary artery bypass grafting. The grafts demonstrated two distinct forms of graft occlusion: four out of the 14 graft occlusions (29%) resulted from severe hyperplastic transformation of the intima complicated by thrombi attached to the degenerating liminal endothelium; the remaining graft occlusions (71%) were due to the development of atherosclerotic lesions associated with mural thrombosis. Hiperplastically altered intimal segments were practically free of neovascularisation while atherosclerotic-like lesions contained neovessels irregularly distributed throughout. Intimal neovessels were located exclusively in microzones enriched with VEGF-expressing cells and, furthermore, neovascular endothelial cells themselves also displayed VEGF immunopositivity. Double-immunostaining revealed that in areas of neovascularisation, the vast majority macrophages (CD68+) expressed VEGF. Some CD68+ foam cells that surrounded branches of neovascularisation were also VEGF-positive. These findings suggest that VEGF expressed by neovascular endothelial cells and by macrophages may act as a local regulator of endothelial cells functions and may induce intimal neovascularisation in aortocoronary saphenous vein bypass grafts affected by atherosclerosis.

Evidence that dendritic cells infiltrate atherosclerotic lesions in apolipoprotein E-deficient mice.

Earlier we reported that atherosclerotic lesions of apoE-deficient mice contained cells which stained positively with anti-S-100 antibody and that cells exhibiting the ultrastructural features of dendritic cells were present in the aortic lesions. These observations suggested that dendritic cells might be involved in mouse atherosclerosis. By employing DEC-205 and MIDC-8 antibodies specific for dendritic cells, the present study has established that dendritic cells indeed accumulate in atherosclerotic lesions of apoE-deficient mice. Finding dendritic cells infiltrating atherosclerotic lesions in apoE-deficient mice offers the possibility of investigating the migratory routes of dendritic cells and their involvement in T-cell activation.

Novel mutations in the SDHD gene in pedigrees with familial carotid body paraganglioma and sensorineural hearing loss.

Paraganglioma (PGL) is a rare disorder characterized by tumors of the head and neck region. Between 10% and 50% of cases of PGL are familial, and the disease is autosomal dominant and subject to age-dependent penetrance and imprinting. The paraganglioma gene (PGL1) has been mapped to 11q22.3-q23, and recently germline mutations in the SDHD gene have been identified. The SDHD region contains another gene, DPP2/TIMM8B, the homolog of which causes dystonia and deafness seen in Mohr-Tranebjaerg syndrome. Using four PGL pedigrees, two of which exhibit coinheritance of PGL and sensorineural hearing loss or tinnitus, analysis of 14 microsatellite markers provided support for linkage to the PGL1 locus. Sequence analysis identified novel mutations in exon 1 and exon 3 of the SDHD gene, including a novel two base pair deletion in exon 3 creating a premature stop codon at position 67; a novel three base pair deletion in exon 3 resulting in the loss of Tyr-93; a missense mutation in exon 3 resulting in the substitution of Leu-81 for Pro-81; and a novel G-to-C substitution in exon 1 resulting in the substitution of Met-1 for Ile-1. No base changes were detected in the DPP2/TIMM8B gene. There was no apparent loss of heterozygosity at the site of the SDHD mutations. However, RT-PCR analysis of tumor samples showed monoallelic expression of the mutant (paternal) allele as expected for imprinting. This has not previously been shown for this disorder. The inheritance and expression of the SDHD gene is consistent with the PGL1 gene being subject to genomic imprinting.

Expression of apoptosis-related proteins and structural features of cell death in explanted aortocoronary saphenous vein bypass grafts.

This study aimed to investigate the features of cell death occurring in aortocoronary saphenous vein bypass grafts. Human aortocoronary saphenous vein bypass grafts with angiographic luminal stenosis of > 75% were explanted from 14 patients at redo coronary artery bypass grafting. Proteins associated with apoptotic pathways were identified immunohistochemically using antibodies to Bcl-2, Fas, BAX, p53 and CPP32. Cells undergoing DNA fragmentation were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). DNA synthesis was investigated using the antibody to proliferating cell nuclear antigen (PCNA). Ultrastructural features of cell death were examined by electron microscopy. Anti-apoptotic (Bcl-2) and pro-apoptotic (Bax, p53, CPP32 and Fas) proteins were expressed throughout the graft wall, but marked differences in the characteristics of cell death were noted between atherosclerotic and non-atherosclerotic areas of the intima. In atherosclerotic areas, pro-apoptotic proteins were widely expressed, but ultrastructural analysis failed to identify cells showing typical features of apoptosis. In these areas, necrotic cells were frequently observed, with negative correlation of Bcl-2 expression with TUNEL. Pro-apoptotic proteins showed no correlation with TUNEL. In contrast, in non-atherosclerotic areas of vein grafts, the expression of both anti-apoptotic (Bcl-2) and pro-apoptotic proteins (p53, Bax and CPP32) correlated with TUNEL. In atherosclerotic areas, non-atherosclerotic intimal areas, and in the underlying media, the numbers of TUNEL+ cells correlated with PCNA positivity. Ultrastructurally, apoptotic bodies and features of necrosis were observed in non-atherosclerotic areas of grafts. The present observations indicate that in atherosclerotic areas, cell death occurs mainly by necrosis, while in non-atherosclerotic areas, cell death occurs by both necrosis and apoptosis. An imbalance between DNA fragmentation and DNA synthesis may contribute to graft instability and failure.

CD40 co-stimulatory molecule expression by dendritic cells in primary atherosclerotic lesions in carotid arteries and in stenotic saphenous vein coronary artery grafts.

We have previously identified dendritic cells in the intima of human large arteries and stenotic vein coronary bypass grafts. The mechanisms by which these dendritic cells might regulate immune responses in atherosclerotic lesions and stenotic vein grafts are unknown. The aim of the present study was to investigate whether dendritic cells in carotid plaques and in stenotic aortocoronary vein grafts express an immunoregulatory molecule CD40. Segments of wall from eight carotid arteries and three stenotic aortocoronary saphenous vein grafts were obtained at operation. CD40+ cells were detected in all specimens of both occluded and stenotic grafts and carotid plaques. Although CD40+ cells of various cell types were intermingled in most areas within the plaques and stenotic grafts, there were sites where CD40+ cells were located in small groups. Consecutive sections demonstrated that a small population of CD40+ cells stained positively for S100. These CD40+/S100+ cells were clustered within the intima but were also found in the media and adventitia. This suggests that dendritic cells, which accumulate within vessels affected by atherosclerosis and graft disease, express CD40 co-stimulatory molecule. The expression of CD40 molecule on the dendritic cells may be important in regulating T cell responses within atherosclerotic plaques and stenotic vein grafts.

Phenotype determination of anti-GM3 positive cells in atherosclerotic lesions of the human aorta. Hypothetical role of ganglioside GM3 in foam cell formation.

Earlier we reported that atherosclerotic plaques contain cells which were specifically and very intensively stained with anti-GM3 antibodies although no GM3 positive cells were detected in the normal non-diseased arterial intima. Because of their lipid inclusions, GM3 positive cells in atherosclerotic lesions seemed to be foam cells but their origin needed clarification. Using an immunohistochemical technique in the present work, we showed that some of these foam cells contained CD68 antigen. However, the most intense accumulation of GM3 occurred in the areas composed of foam cells which did not stain with any cell type-specific antibodies, including antibodies to macrophages (anti-CD68) and smooth muscle cells (anti-smooth muscle alpha-actin), perhaps, because the cell type-specific antigens were lost during the transformation of intimal cells into foam cells. Ultrastructural analysis of the areas where foam cells overexpressed GM3 demonstrated that some foam cells lacked both a basal membrane and myofilaments but contained a large number of secondary lysosomes and phagolysosomes, morphological features which might indicate their macrophage origin. Other foam cells contained a few myofilaments and fragments of basal membrane around their plasmalemmal membrane, suggesting a smooth muscle cell origin. These observations indicate that accumulation of excessive amounts of GM3 occurs in different cell types transforming into foam cells. We suggest that up-regulation of GM3 synthesis in intimal cells might be an essential event in foam cell formation. Shedding of a large number of membrane-bound microvesicles from the cell surface of foam cells was observed in areas of atherosclerotic lesions corresponding to extracellular GM3 accumulation. We speculate that extracellularly localised GM3 might affect the differentiation and modification of intimal cells in atherosclerotic lesions.

Colonisation of prosthetic grafts by immunocompetent cells in a sheep model.

The present study examined the distribution of immunocompetent cells in synthetic vascular grafts in an experimental sheep model. Sixty-two adult Merino sheep underwent synthetic patch closure of a longitudinal arteriotomy in the left common carotid artery. The synthetic patch materials used were gelatin sealed Dacron (n=10), fluoropassivated Dacron (n=10), Fluoropassiv (n=12), polyurethane (n=10), expanded polytetrafluoroethylene (n=10) and carbon-lined expanded polytetrafluoroethylene (n=10). The sheep were sacrificed after four weeks when the prosthetic patches were harvested and fixed in 10% neutral buffered formalin. Transverse sections were taken along the graft and paraffin embedded. Serial sections were stained with cell type specific antibodies to identify T-lymphocytes (CD3(+)), dendritic cells (S-100(+)), endothelial cells (von Willebrand factor(+)) and smooth muscle cells (smooth muscle alpha-actin(+)). All six graft types contained CD3(+) and S-100(+) cells in the neointima, within the synthetic matrix and in the perigraft layer. Three different tissue responses to synthetic materials were observed and the grafts were classified accordingly into three groups: (1) gelatin sealed Dacron, fluoropassivated Dacron and Fluoropassiv; (2) expanded polytetrafluoroethylene and carbon-lined expanded polytetrafluoroethylene; (3) polyurethane. The three synthetic materials in Group 1 showed almost identical reactions with least accumulation of immunocompetent cells within the synthetic material but greater accumulation of immuno-inflammatory infiltrates in the perigraft vascular tissue. In this group, new vessels penetrated into the synthetic material and there was prominent formation of foreign body (giant) cells. Group 2 showed greater accumulation of immunocompetent cells within the synthetic material itself but only sparse immuno-inflammatory infiltrates in the perigraft tissue. Group 3 showed a high degree of inflammatory response within both the synthetic material and the perigraft vascular tissue. These observations demonstrate that immunocompetent cells colonise the synthetic matrix of grafts and accumulate in the perigraft tissue, but inflammatory responses vary in different graft types.

Immunohistochemical and ultrastructural evidence that dendritic cells infiltrate stenotic aortocoronary saphenous vein bypass grafts.

We earlier speculated that antigen-presenting dendritic cells may be involved in the immune reactions leading to saphenous vein bypass graft failure. The purpose of this study was to confirm whether dendritic cells are present in stenotic human saphenous vein bypass grafts. Segments of stenotic saphenous vein grafts were explanted from 14 patients at re-do bypass operation and ten normal saphenous veins were harvested during femoro-popliteal grafting. Sections of specimens were analysed using cell type specific antibodies to identify dendritic cells (CD1a, S-100), T-lymphocytes (CD3), macrophages (CD68), smooth muscle cells (alpha-SMA) and endothelial cells (FVIII). Dual immunostaining, confocal immunofluorescent laser scanning microscopy and electron microscopy were used. Stenotic grafts showed structural alterations of intimal hyperplasia and varying degrees of atherosclerotic degeneration. No cells expressing CD1a and S-100 were observed in the intima and media of normal saphenous veins. Cells expressing these antigens were present around areas of medial neovascularization and within intimal atherosclerotic lesions in saphenous vein bypass grafts. Electron microscopy demonstrated the presence of cells containing a well-developed tubulovesicular system which is unique to cells from the dendritic cell family. Double immunohistochemistry and confocal immunofluorescent microscopy revealed the co-localization of T-lymphocytes with dendritic cells. Dendritic cells are present in stenotic saphenous vein bypass grafts. Dendritic cells may be responsible for antigen presentation and modulation of immune reactions in accelerated graft atherosclerosis through their interaction with T-lymphocytes.

Documented prevalence of HIV and hepatitis C infection in patients with penetrating trauma.

BACKGROUND: Trauma patients infected with human immunodeficiency virus (HIV) or hepatitis C (HCV) pose specific problems to health-care workers due to the risk of exposure to these agents in blood and other body fluids. Studies of patients with penetrating trauma in the USA have shown a higher prevalence of HIV and HCV infection than the general population. No studies have examined the prevalence of these infections in Australian trauma patients. METHODS: The medical records of all patients presenting to St Vincent's Hospital, Sydney, from January 1994 to December 1998, with a stab wound to the neck, chest or abdomen, or with a gunshot wound to any anatomical site, were retrospectively reviewed. The number of patients with a history of HIV or HCV infection, or with risk factors for these such as male-to-male sexual intercourse and intravenous drug use, were recorded. RESULTS: The medical records of 148 patients with stab wounds to the neck, chest or abdomen, or with gunshot wounds were examined. Risk factors for HIV or HCV infection were recorded in 31 patients (21%). Two patients (1.3%) had a history of HIV infection and a further eight patients (5.4%) were known to have HCV. CONCLUSIONS: There was a high prevalence of risk factors for HIV and HCV in patients with major penetrating wounds at St Vincent's Hospital. The prevalence of documented HIV and HCV infection was subsequently greater than that expected in the general population, highlighting the risks to health-care workers managing these patients.

Incidence of acquired immune deficiency syndrome (AIDS)-related disorders at laparotomy in patients with AIDS.

BACKGROUND: Patients with acquired immune deficiency syndrome (AIDS) present for surgical management of abdominal conditions that are complications of advanced human immunodeficiency virus (HIV) infection or that are caused by other disease not related to AIDS. This study compared the clinical details and postoperative outcomes of patients with AIDS-related diseases found at laparotomy with those of patients with non-AIDS-related disorders. METHODS: The medical records of 30 consecutive patients with AIDS who underwent laparotomy were examined retrospectively. RESULTS: Fourteen patients had AIDS-related pathologies found at laparotomy, 13 had disease processes that were not AIDS related and three had no abnormal findings at laparotomy. Patients with AIDS-related conditions at laparotomy had lower mean body-weight, serum albumin concentration and CD4 lymphocyte count, and required a longer hospital admission than those who had non-AIDS-related disease. The duration of HIV infection and the number of complications and deaths were similar in the two groups. Complications occurred in 21 patients and there were five deaths (30-day mortality rate 17 per cent). CONCLUSION: AIDS-related pathologies are commonly found at laparotomy in patients with HIV/AIDS. An AIDS-related diagnosis does not confer a greater risk of complication or postoperative death than other conditions.

Vascular-associated lymphoid tissue (VALT) involvement in aortic aneurysm.

Vascular-associated lymphoid tissue (VALT) consisting of accumulations of immunocompetent and antigen presenting cells has recently been recognised in the arterial wall. In this study, we investigated the involvement of VALT in immune responses in abdominal aortic aneurysms (AAAs). Tissue samples were collected during operations from 31 patients with atherosclerotic infrarenal abdominal aortic aneurysms ranging in diameters from 5 to 8 cm. The specimens were immediately frozen and examined using single and double immunohistochemical staining. T-cell subpopulations, B-cells, dendritic cells and macrophages were identified using cell type specific antibodies. Cell contacts were examined by electron microscopy. Most inflammatory infiltrates were found in the adventitia. T-cells were the predominant cell type in a majority of inflammatory infiltrates but in seventeen cases, typical lymphoid follicles with B-cells forming germinative centres were also observed. In eight cases, the lymphoid follicles aggregated in lymph node-like structures. Dendritic cells were present within all inflammatory infiltrates and contacted lymphocytes. The present observations show that in aortic aneurysm, VALT is involved in immune responses and its activation mostly occurs in the adventitia. The formation of lymphoid follicles and lymph node-like structures in the adventitia suggests that VALT might locally serve the entire complex of both cellular and humoral immune responses in the aneurysmal wall.