Sudden, unexpected cardiac or unexplained death in England: a national survey.

BACKGROUND: Post-mortem examinations of adults who were apparently healthy but died suddenly and unexpectedly sometimes reveal no morphological abnormalities to explain their deaths. The frequency of such unexplained deaths in relation to other causes of sudden cardiac death is not known. AIM: To estimate the frequency of sudden unexpected cardiac or unexplained death in England. DESIGN: Prospective survey using a stratified random sample of 83 of the 132 H.M. Coroner's jurisdictions in England. METHODS: Consecutive White Caucasians, aged 16-64 years, with no medical history of cardiac disease, seen alive within 12 h of death, on whom autopsy found either a cardiac or no identifiable cause of death, were included. The coroner's officer sent a copy of the post-mortem report and a completed case registration form to the investigators, with tissue samples. RESULTS: Sixty-seven (81%) coroners participated, each maintaining prospective surveillance for 4 months. Of 692 ascertained cases, case registration forms were received for 650 (94%), post-mortem reports for 682 (99%), blood samples for 569 (82%), myocardial slices for 517 (75%) and whole hearts for 47 (7%). In cases with myocardial tissue, death was ascribed to ischaemic heart disease in 465 (82.4%). In 43.1% the ischaemia was acute, in 19.1% there was myocardial scarring but no acute ischaemia, and 20.2% had coronary atheroma only. Death was due to left ventricular hypertrophy in 32 (5.7%), to other cardiac causes in 30 (5.3%) and in 23 (4.1%) there was no clear cause. Those with cardiac causes were 81% male, median ages 55.9 (male) and 56.6 (female) years. The 23 unexplained deaths were 57% female, median ages 40.5 (male) and 54.9 (female) years. The estimated annual frequency of sudden unexpected death due to cardiac or unidentified causes, in English adults of employment age, was 11/100,000 (3481 annual deaths). DISCUSSION: In 4.1% of sudden unexpected deaths under 65 years, no cause was found. Until it becomes accepted practice to identify these cases by a name, such as Sudden Adult Death Syndrome (SADS), it will not be possible to study their aetiology systematically.

Microsatellite mutation of type II transforming growth factor-beta receptor is rare in atherosclerotic plaques.

A somatic mutation within a microsatellite polyA tract in the coding region of the type II transforming growth factor (TGF)-beta receptor gene was reported to occur in human atherosclerotic and restenotic lesions. This mutation occurs frequently in colorectal cancer with the replication error repair phenotype and results in loss of sensitivity to the growth inhibitory effects of TGF-beta in cells from the tumors. The mutation was proposed to account for the clonal expansion of vascular smooth muscle cells observed in atherosclerotic plaques, through loss of the growth inhibitory effect of TGF-beta. The frequency of the mutation and the extent of clonal expansion of the mutated cells have major implications for the mechanism of atherogenesis and therapeutic strategies. We analyzed a set of 22 coronary arterial and 9 aortic samples containing early to advanced atherosclerotic lesions for the mutation in the type II TGF-beta receptor polyA tract. Only 1 coronary arterial sample from an advanced lesion showed detectable amounts of the mutation, present at a low level (8% of the DNA sample). The data imply that the mutation occurs only at low frequency and is not a major mechanistic contributor to the development of atherosclerosis.

A potential role for sterol 27-hydroxylase in atherogenesis.

OBJECTIVE: 27-hydroxycholesterol is the product of the mitochondrial cytochrome P450 sterol 27-hydroxylase, a key enzyme in cholesterol metabolism present in most tissues of the body. 27-hydroxycholesterol increases in abundance with progression of human atherosclerotic lesions, therefore the aim of this study was to determine the pattern of sterol 27-hydroxylase gene expression in normal and diseased arteries and to identify the cell types responsible for its expression. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridisation, utilising a sterol 27-hydroxylase cDNA probe, and immunohistochemistry, utilising an antibody to sterol 27-hydroxylase, together with an antibody to smooth muscle cell alpha-actin and an antibody to CD68, a marker for macrophages, were used to study expression of 27-hydroxylase in arterial specimens. In addition, RT-PCR was used to study expression of 27-hydroxylase in cultured macrophages and smooth muscle cells. RESULTS: Semi-quantitative RT-PCR analysis of normal and atherosclerotic human aortas showed that 27-hydroxylase is constitutively expressed in the normal artery wall, and is substantially up-regulated in atherosclerosis. RT-PCR analysis of 27-hydroxylase expression in vitro demonstrated that macrophages constitutively express high levels throughout their differentiation in culture whilst de-differentiated vascular smooth muscle cells express very low levels. In situ hybridisation revealed that in normal artery and fatty streaks, expression of mRNA for 27-hydroxylase was low in the media, but higher in intimal smooth muscle cells. The macrophages of fatty streaks expressed low or undetectable levels of 27-hydroxylase. However in advanced lesions the highest expression of 27-hydroxylase was detectable in macrophages. Immunohistochemistry demonstrated that high levels of 27-hydroxylase protein occurred in macrophages near the shoulder region of plaques, at the edge of the lipid core. CONCLUSIONS: 27-hydroxylase may constitute a protective mechanism for removing cholesterol from macrophages and smooth muscle cells. Genetic heterogeneity resulting in differences in sterol 27-hydroxylase activity between individuals may affect their ability to deal with accumulated cholesterol in the arterial intima, and hence their relative degree of predisposition to atherosclerosis.

Sensitivity to Fas-mediated apoptosis is determined below receptor level in human vascular smooth muscle cells.

Despite Fas expression, many cells resist Fas-induced apoptosis. Although differences in surface Fas expression can explain Fas resistance, multiple proteins below receptor level also inhibit Fas-induced apoptosis. To examine the mechanism of Fas resistance, we studied Fas-induced apoptosis in human medial vascular smooth muscle cells (VSMCs) from healthy coronary arteries. VSMCs showed marked heterogeneity to Fas-induced apoptosis, exhibiting both Fas-resistant (98.1+/-2.3% viable, n = 4, P = NS) and Fas-sensitive (31.3+/-2.6% viable, n = 3, P<0.01) cells. Fas-resistant VSMCs expressed surface Fas and could recruit RIP, indicating that functional receptor complexes were formed. However, Fas-resistant cells showed reduced expression of FADD, Fas ligand, and caspases 3, 7, and 8 and increased expression of FLIP and c-IAP-1. Fas-induced apoptosis was associated with cleavage of caspase 3 and blocked by inhibitors of caspase 3 or 8 but not caspase 1, 6, or 7. Selective inhibition of caspase 3 or 8 by antisense transfection inhibited Fas-induced apoptosis, but their reexpression could not rescue the Fas-resistant phenotype. In vivo, medial VSMCs showed marked heterogeneity of expression of caspase 3. We conclude that Fas sensitivity is determined not only by expression of surface Fas but by differential expression of Fas-signaling proteins below receptor level. Subpopulations of cells within the same tissue have different sensitivities to apoptosis, determined by expression of specific death-signaling proteins.

Expression of mineralisation-regulating proteins in association with human vascular calcification.

OBJECTIVES: These studies aim to investigate the expression and function of mineralisation-regulating proteins in association with human vascular calcification focussing on the similarities and differences between the two major calcification pathologies in man: atherosclerotic, intimal calcification and Monckeberg's sclerotic medial calcification. BACKGROUND: A number of studies have documented expression of mineralisation-regulating proteins in association with human atherosclerotic calcification leading to the suggestion that human vascular calcification may be a regulated process with similarities to developmental osteogenesis. METHODS: In situ hybridisation, immunohistochemistry and semi-quantitative RT-PCR analysis were used to determine the temporal and spatial expression patterns of mineralisation-regulating proteins within human calcified vascular lesions. Additionally, the expression and regulation of bone-associated proteins was analysed during spontaneous calcification of human VSMCs in vitro. RESULTS: In association with both medial and intimal calcification, the temporal changes in expression of mineralisation-regulating proteins are similar. Some constitutively expressed bone-associated proteins, including matrix Gla protein (MGP), are down-regulated in association with calcification while expression of a number of bone-associated proteins, not normally expressed in the vessel wall, are induced including alkaline phosphatase (ALK), bone sialoprotein (BSP) and bone Gla protein (BGP). In medial calcification the source of expression of these mineralisation-regulating proteins is VSMCs while in intimal lesions both VSMCs and macrophages express them. Furthermore, these bone-associated proteins are spontaneously expressed by VSMCs in vitro suggesting that human VSMCs are capable of simultaneously exhibiting smooth muscle and osteogenic-like properties. CONCLUSIONS: These studies imply that both medial and intimal vascular calcification are regulated processes; however the aetiology of each pathology differs.

Medial localization of mineralization-regulating proteins in association with Mönckeberg's sclerosis: evidence for smooth muscle cell-mediated vascular calcification.

BACKGROUND: Calcification of the media of peripheral arteries is referred to as Mönckeberg's sclerosis (MS) and occurs commonly in aged and diabetic individuals. Its pathogenesis is unknown, but its presence predicts risk of cardiovascular events and leg amputation in diabetic patients. Several studies have documented expression of bone-associated genes in association with intimal atherosclerotic calcification, leading to the suggestion that vascular calcification may be a regulated process with similarities to developmental osteogenesis. Therefore, we examined gene expression in vessels with MS to determine whether there was evidence for a regulated calcification process in the vessel media. METHODS AND RESULTS: In situ hybridization, immunohistochemistry, and semiquantitative reverse-transcription polymerase chain reaction were used to examine the expression of mineralization-regulating proteins in human peripheral arteries with and without MS. MS occurred in direct apposition to medial vascular smooth muscle cells (VSMCs) in the absence of macrophages or lipid. These VSMCs expressed the smooth muscle-specific gene SM22alpha and high levels of matrix Gla protein but little osteopontin mRNA. Compared with normal vessels, vessels with MS globally expressed lower levels of matrix Gla protein and osteonectin, whereas alkaline phosphatase, bone sialoprotein, bone Gla protein, and collagen II, all indicators of osteogenesis/chondrogenesis, were upregulated. Furthermore, VSMCs derived from MS lesions exhibited osteoblastic properties and mineralized in vitro. CONCLUSIONS: These data indicate that medial calcification in MS lesions is an active process potentially orchestrated by phenotypically modified VSMCs.

Aquaporin-1 is expressed by vascular smooth muscle cells and mediates rapid water transport across vascular cell membranes.

The aquaporins are a rapidly expanding family of highly conserved proteins which function as transmembrane water channels. We have previously shown that the gene for aquaporin-1 (AQP-1) is expressed in rat, aortic vascular smooth muscle cells (VSMCs) implying a specific role for AQP-1 in vascular function. In this study we set out to document the expression of AQP-1 in human arteries and found mRNA and protein in normal endothelial and VSMCs of human arteries and capillaries and in a subset of VSMCs in human atherosclerotic plaques. Secondly, we examined the regulation of AQP-1 gene expression during vascular development and following vascular injury. Studies in the rat demonstrated that AQP-1 mRNA is induced in the neonatal aorta at week 2 of postnatal development and that the protein is present in neointimal VSMCs following balloon injury. Finally, by measuring the rate of change in cell size induced by changes in external osmolarity and demonstrating that water transport can be inhibited with mercuric chloride, we show that AQP-1 is responsible for water transport across human VSMC membranes. Thus, this study provides evidence for a hitherto unrecognised role for aquaporins in mediating rapid water transport across human VSMC membranes. By analogy with other tissues, these data argue for an important role for AQP-1 in regulating transcellular fluid flow and tissue hydration.

Changes in elemental concentrations are associated with early stages of apoptosis in human monocyte-macrophages exposed to oxidized low-density lipoprotein: an X-ray microanalytical study.

This study examines ion homeostasis in monocyte-macrophages committed to death by apoptosis. X-ray microanalysis has been used to demonstrate that intracellular concentrations of potassium decreased whilst those of sodium increased following 3 h of exposure to 100 microg/ml of oxidized low-density lipoprotein (LDL) in vitro. In contrast, the maximal incidence of cell death, as determined by the inability to exclude trypan blue, was not seen until 24 h of exposure. At 12 h, less than 1 per cent of cells were stained using terminal transferase-mediated DNA nick-end labelling, which is generally accepted as a marker of late stages in the apoptotic pathway. This is the first demonstration of early perturbations of ion homeostasis in monocyte-macrophages exposed to concentrations of oxidized LDL known to cause apoptosis.

Cytochemical demonstration of sites of hydrogen peroxide generation and increased vascular permeability in isolated pig hearts after ischaemia and reperfusion.

Isolated pig hearts, subsequently perfused with pig or human blood, were prepared for the cytochemical demonstration of sites of hydrogen peroxide generation and increased vascular permeability. Oxidant stress was associated with ultrastructural changes commonly seen following myocardial reperfusion. In addition, the precipitation of cerium perhydroxide following perfusion with physiological saline containing cerium chloride suggested the vascular endothelium and leukocytes as sources of oxidants. This was associated with rapid penetration of horseradish peroxidase through the intercellular clefts of the vascular endothelium into the interstitial space, suggesting increased vascular leakiness at these sites. The rapid penetration of horseradish peroxidase was observed at all monitored periods of reperfusion with pig or human blood. This indicates that the increased permeability occurred during the ischaemic period and continued during reperfusion. Morphological damage was greatest in pig hearts reperfused with whole human blood and this was attenuated if the blood was preabsorbed to remove antibodies prior to reperfusion. We conclude that oxidant stress was initiated during ischaemia and continued during reperfusion in this model.

Identification of osteoglycin as a component of the vascular matrix. Differential expression by vascular smooth muscle cells during neointima formation and in atherosclerotic plaques.

Using differential cDNA screening, we demonstrated that the bone-associated glycoprotein osteoglycin was highly expressed in differentiated adult rat vascular smooth muscle cells (VSMCs) but downregulated in VSMCs that had undergone proliferation in vitro. Further experiments in vitro revealed that osteoglycin gene expression was downregulated by a number of cytokines expressed in vivo (often in association with vascular injury) including basic fibroblast growth factor, transforming growth factor-beta, platelet-derived growth factor, and angiotensin II. In the normal adult rat carotid artery, osteoglycin was expressed in both the media and adventitia. However, osteoglycin mRNA expression was substantially increased in the adventitia and neointima 14 days after balloon injury, implying a role for this protein in vessel remodeling. Northern analysis of mRNA from neonatal rat aortas demonstrated upregulation of osteoglycin mRNA at week 2, after VSMC proliferation had ceased and when matrix modeling was maximal. In situ hybridization studies in human coronary arteries showed that osteoglycin mRNA was expressed by normal medial VSMCs but was downregulated in a subset of intimal VSMCs. Osteoglycin was not expressed in the VSMCs of adventitial vessels but was expressed in a subset of adventitial cells. This expression pattern contrasted with that of SM22 alpha, a contractile protein marker of VSMC differentiation, which was highly expressed in the media of all vessels. These data indicate that osteoglycin is a new marker of differentiated VSMCs and may be an essential component of the normal vascular matrix.

Human leukocyte antigen compatibility in heart transplantation: evidence for a differential role of HLA matching on short- and medium-term patient survival.

BACKGROUND: Studies of the influence of human leukocyte antigen (HLA) matching on cardiac transplant outcome have proved inconclusive, mainly due to the lack of well-matched grafts. However, a growing number of studies report improved clinical course and patient survival in cases with increased HLA compatibility. Opelz et al. believe these benefits justify the introduction of prospective HLA-matching strategies. METHODS: We performed univariate and multivariate analyses to examine the short- and medium-term influence of HLA matching on 556 consecutive primary heart transplants performed at a single center between 1983 and 1994. Overall graft survival at 1, 3, and 5 years was 80%, 74%, and 67% respectively. Sixteen (2.9%) grafts failed within 5 days and were not considered in the analysis of the HLA matching and graft survival data. RESULTS: Complete HLA-A, -B, and -DR typing data were available on 477 transplant pairs. The results demonstrate a 12% 1-year survival advantage for 31 patients with zero to two HLA antigen mismatches compared with three to six mismatches. The influence of each individual locus was 6.1%, 8.4%, and 5.4% for zero HLA-A, -B, and -DR mismatches, respectively, compared with two mismatches. However, when outcome from 1 to 5 years was considered, analysis of the role of each locus revealed marked differences. HLAA-matched grafts (n=45) had a 24% lower survival rate compared with two-antigen-mismatched grafts (n=148; 88% [SE 3.1] vs. 64% [SE 8.2], respectively; P=0.009). Furthermore, 34% of HLA-A-matched grafts failed between 1 and 5 years, compared with only 5% of HLA-B-matched grafts (P=0.013). CONCLUSIONS: These data suggest that although HLA matching is effective at reducing acute graft loss, in the longer term, HLA-A matching may impair survival. HLA-A may serve as a restriction element for indirect presentation of allopeptides or tissue-specific minor histocompatibility antigens, facilitating chronic graft loss. Therefore, we advocate a differential role for HLA matching over two epochs. A blanket approach to prospective matching for heart transplants may be premature for optimal long-term survival.

Polyubiquitin is a new phenotypic marker of contractile vascular smooth muscle cells.

OBJECTIVE: Medial vascular smooth muscle cells (VSMCs) in healthy vessels are phenotypically distinct from their intimal counterparts in vascular disease. To compare the genes expressed in these phenotypes we have previously performed a differential cDNA library screen on cultured rat VSMCs. The aim of this study was to identify and characterise a 2.8 kb transcript, 2E10, which was highly expressed in freshly dispersed rat aortic VSMCs and downregulated in multiply passaged cultured VSMCs. METHODS: Sequence analysis was used to identify the 2.8 kb rat cDNA. After trypsinisation of proliferating cultured rat and human VSMCs, or enzymatic digestion of aortic tunica media, total cytoplasmic RNA was isolated from VSMCs by lysis in Nonidet P-40 and extraction in phenol; 15 micrograms of total cytoplasmic RNA was used in Northern blot analysis with a 32P-[dCTP]-labelled 2E10 cDNA probe. 35S-[dATP]-labelled 2E10 riboprobe was hybridised in situ to frozen sections of normal and diseased human coronary arteries. RESULTS: DNA sequencing identified 2E10 as a rat polyubiquitin which is homologous to the human polyubiquitin, UbC. Northern blot analysis showed that this polyubiquitin was more highly expressed in differentiated, freshly dispersed rat and human aortic VSMCs compared with their dedifferentiated proliferating counterparts. This also identified a 3.2 kb transcript cross-reacting with the polyubiquitin probe which is specific to differentiated rat VSMCs only. However, expression in growth arrested and proliferating VSMCs was identical, suggesting that UbC does not have a role in VSMC growth arrest. In situ hybridisation of the polyubiquitin riboprobe to sections of diseased human coronary arteries indicated much higher expression in medial than in intimal VSMCs. Northern blot analysis of RNA from the developing rat aorta showed that polyubiquitin expression increased substantially after week 2 of neonatal life, coincident with expression of VSMC-specific contractile proteins. CONCLUSIONS: The greater expression of a UbC polyubiquitin transcript in contractile, differentiated VSMCs compared with proliferating, synthetic VSMCs provides a new gene marker for the phenotypic characterisation of VSMCs in vivo. This, and the finding that the developmental induction of expression of polyubiquitin (UbC) mirrors that of VSMC contractile proteins, suggests that ubiquitin, a protein known to associate with and degrade contractile proteins in skeletal muscle, is involved in the function or maintenance of the contractile phenotype of VSMCs.

Foam cell apoptosis and the development of the lipid core of human atherosclerosis.

A characteristic feature of the advanced atherosclerotic lesion is the acellular lipid core, which appears to result at least partly from the death of macrophage foam cells. This study shows that foam cell death at the edge of the lipid core includes both necrosis and apoptosis and that remnants of apoptotic nuclei are present within the lipid core. Apoptotic cells were identified by transmission electron microscopy and by nick end-labelling using terminal deoxynucleotidyl transferase (TUNEL). Some TUNEL-positive cells also expressed proliferating cell nuclear antigen (PCNA). The cause of foam cell death in atherogenesis is unknown, but oxidized low-density lipoprotein (LDL) can cause macrophage apoptosis in vitro and might therefore play a role in the formation and enlargement of the lipid core.

Endothelin peptide and receptors in human atherosclerotic coronary artery and aorta.

The aim of this study was to determine whether there is an alteration in the distribution or quantity of endothelin (ET) peptide or receptor subtypes in human atherosclerotic arteries. Levels of endogenous ET and big ET-1 detectable by radioimmunoassay in human aorta containing raised atherosclerotic plaques were significantly higher than those in histologically normal tissue (Student's t test, P < .01). Immunohistochemistry revealed ET-like immunoreactivity in endothelial cells lining the vessel lumen, neovascularization, recanalization of organized thrombus, and regions rich in macrophages. Little immunoreactivity was associated with vascular smooth muscle cells (VSMCs). Saturation binding assays with [125I]ET-1 indicated comparable affinities and maximal densities of receptors in the media of diseased and normal coronary arteries. Quantitative autoradiography with subtype-selective radioligands revealed similar small proportions of ETB receptors in the diseased and normal arterial media. In arteries with early and late disease, ETA receptors were localized to medial smooth muscle but were lacking in the VSMCs of the intimal layer, where ETB receptors were absent. ETB receptors were detected on perivascular nerves and lymphoid aggregates. In atherosclerotic arteries, microautoradiography localized ETB receptors to neovascularization and, interestingly, to macrophages. The results of this study indicate that there is an increase in ET and big ET-1 associated with fully developed atherosclerotic plaques. It is likely that this is derived from endothelial cells and macrophages but not VSMCs. ETA receptors predominate in the media of both normal and diseased arteries. ET receptors are deficient in intimal smooth muscle, and ETB receptors, where present, are found on endothelial cells and macrophages.

Serological and molecular evidence of enterovirus infection in patients with end-stage dilated cardiomyopathy.

OBJECTIVE: To study the relative diagnostic value of enterovirus-specific molecular biological and serological assays in patients with end-stage dilated cardiomyopathy, and to investigate the possible role of other cardiotropic viruses in dilated cardiomyopathy. DESIGN: Analysis of recipient myocardial tissue and serum from patients with dilated cardiomyopathy and controls undergoing cardiac transplantation for end-stage cardiac disease. SETTING: University virology department and transplantation unit. METHODS: Reverse transcriptase-polymerase chain reaction and nucleotide sequence analysis of myocardial RNA and DNA; enterovirus-specific in situ hybridization; enterovirus-specific immunoglobulin M detection. RESULTS: Enterovirus RNA was detected in myocardial tissue from only a small proportion of (five of 75) hearts. However, although enterovirus-specific immunoglobulin M responses were detected in 22 (28%) of 39 controls patients, a significantly higher prevalence was observed among patients with dilated cardiomyopathy (22 (56%) of 39 patients; P < 0.005). All enteroviruses detected in myocardium showed greatest nucleotide sequence homology with coxsackievirus type B3. Detection of enterovirus RNA in myocardium by the polymerase chain reaction and by in situ hybridisation gave comparable results. Other potentially cardiotropic virus genomes, including human cytomegalovirus, influenzaviruses, and coronaviruses were not detected in myocardium. CONCLUSION: This study found that enterovirus-specific immunoglobulin M responses provided the strongest evidence of enterovirus involvement in patients with end-stage dilated cardiomyopathy. However, the high background prevalence of these responses limits their diagnostic value. The finding that enteroviruses detected in myocardium were coxsackievirus type B3 accords with recent findings in patients with acute myocarditis, and indicates that this serotype is the major cardiotropic human enterovirus.

Tissue expression of human complement inhibitor, decay-accelerating factor, in transgenic pigs. A potential approach for preventing xenograft rejection.

Since complement-mediated hyperacute rejection of xenografts prevents the use of pigs as organ donors to man, the development of transgenic animals expressing species-specific complement inhibitors could provide a strategy for overcoming hyperacute rejection. The complement inhibitor, human decay-accelerating factor (hDAF), prevents the assembly of C3 and C5 convertases. In this article, the first histologic analysis of hDAF expression in pig tissues, specifically expression in endothelial cells of pigs transgenic for hDAF, is described. Twenty-seven transgenic pigs were categorized into 4 groups based on the expression patterns in endothelial, vascular smooth muscle, and squamous epithelial cells of skin biopsy specimens. Skin biopsy specimens permitted evaluation of the pigs without the need to kill them or to perform invasive procedures. Sixteen cases demonstrated endothelial cell staining. Complete necropsy evaluation, available in 14 of the 27 pigs, correlated with the skin biopsy specimen expression of hDAF. The immunoperoxidase data matched identically with the presence of the mRNA transcript in 25 of the 26 cases where RNA data were available. Also, the staining patterns of 6 transgenic pig founders and their 9 offspring (total of 9 founder-offspring pairs) correlated. Since transgenes are variably expressed in different cell types and since tissue lysates represent a melange of cell types, histologic evaluation for protein expression in tissues from transgenic animals will be critical if they are to be bred to become clinical organ donors. In addition to endothelial expression of hDAF, its expression on vascular smooth muscle cells may be important in preventing tissue damage when breaks in the endothelium occur.

Evidence that the death of macrophage foam cells contributes to the lipid core of atheroma.

Sections of human atherosclerotic lesions of different stages show that, in early lesions, the acellular lipid core is usually immediately adjacent to the deepest edge of a collection of macrophage foam cells. Advanced lesions with a large lipid core have variable numbers of macrophage foam cells, close to the lateral edges, or shoulders, of the core. In both early and advanced lesions, some of the macrophages nearest the core appear to be dying. Lipid cores contain two materials which in earlier lesions are found only in macrophages, namely ceroid and CD68 antigen, but do not contain recognisable smooth muscle cell actin. It is concluded that death of macrophage foam cells contributes to the origin and slow enlargement of the lipid core. The cause of macrophage death is not yet certain, but is under investigation.

Gene expression and vascular smooth muscle cell phenotype.

Vascular smooth muscle cells (VSMCs) are involved in a number of vascular disease processes including hypertension and atherosclerosis. However, their role in the pathogenesis of vascular disease is largely undetermined. We and others have studied rat VSMCs in cell culture as a model for VSMC behaviour in vivo. In recent experiments we have applied molecular biological techniques to compare genes expressed by normal contractile VSMCs with those expressed by VSMCs which have undergone several passages in cell culture. Using differential screening of a cDNA library derived from cultured rat aortic VSMC RNA we identified seven genes which are preferentially expressed by contractile VSMCs; alpha-smooth muscle actin, gamma-smooth muscle actin, calponin, phospholamban, tropoelastin, SM22 alpha and CHIP28, and two which are preferentially expressed in passaged cells which have down-regulated their contractile proteins; osteopontin (OP) and matrix Gla protein (MGP). In situ hybridization studies have confirmed that calponin and SM22 alpha, are highly expressed by medial VSMCs in human coronary arteries with little or no expression in the atheromatous intima whilst the converse is true for OP and MGP. Studies by ourselves and others have confirmed that OP is a marker for proliferating rat VSMCs both in vitro and in vivo. However, the evidence that OP is expressed by proliferating human VSMCs is less convincing.

Distribution of endothelin receptors in atherosclerotic human coronary arteries.

It has been suggested that the potent vasoconstrictor and proliferative agent endothelin (ET) may contribute to the development and effects of atherosclerosis. The aim of this study was to use autoradiography to examine the distribution of ET receptors in human coronary artery with a range of pre-atherosclerotic and atherosclerotic lesions. ET-1 receptors in epicardial coronary artery sections were visualized according to the binding of [125I]ET-1 (100 pM), the ETA-selective radioligand [125I]PD151242 (100 pM), and the ETB-selective [125I]BQ3020 (300 pM). Dense [125I]ET-1 binding was demonstrated in the smooth muscle of the media of all arteries studied and ETA receptors, defined by [125I]PD151242 binding, predominated. Intense [125I]BQ3020 binding to ETB receptors was apparent on perivascular structures, such as adventitial lymphatics. In contrast, ET receptors were absent in neointimal smooth muscle. In regions of recanalized organized thrombus, microautoradiography revealed ETA receptors on the smooth muscle of new vessels. Discrete clusters of ETB receptors were detected in sections through the atherosclerotic arterial wall. A similar pattern of staining for von Willebrand factor was seen in adjacent sections. This suggests that ETB receptors are present on endothelial cells of neovascularization, penetrating the diseased media.