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N J Severs

Publications and source records attributed to N J Severs.

At least 73 records · Page 4Linked to original sources
PubMed1996

Altered patterns of cardiac intercellular junction distribution in hypertrophic cardiomyopathy.

OBJECTIVE: To examine the distribution pattern of intercellular junctions (the mechanically coupling desmosomes and the electrically coupling gap junctions) in hypertrophic cardiomyopathy (HCM) hearts showing myofibre disarray. DESIGN: Samples from six necropsied hearts were studied, representing the interventricular septum and the free walls of the left and right ventricles. Immunohistochemical labelling of desmoplakin was used as a marker for desmosomes, and of connexin43 as a marker for gap junctions, in single and double stainings. The slides were examined by confocal laser scanning microscopy. RESULTS: Marked disorganisation of intercalated discs was observed in areas featuring myofibre disarray. Besides overall derangement, localised abnormalities in desmosome organisation were evident, which included: (1) the formation of abnormally enlarged megadiscs; (2) the presence of intersecting disc structures; and (3) aberrant side to side desmosomal connections. Gap junctional abnormalities included: (1) random distribution of gap junctions over the surface of myocytes, rather than localisation to intercalated discs; (2) abundant side to side gap junction connections between adjacent myocytes; and (3) formation of abnormally shaped gap junctions. Circles of myocytes continuously interconnected by gap junctions were also observed. Regions of the diseased hearts lacking myofibre disarray, and control hearts of normal patients and patients with other cardiac diseases, did not show these alterations. CONCLUSIONS: The disorganisation of the intercellular junctions associated with myofibre disarray in HCM may play an important role in the pathophysiological manifestations of the disease. The remodelling of gap junction distribution may underlie the formation of an arrhythmogenic substrate, thereby contributing to the generation and maintenance of cardiac arrhythmias associated with HCM.

Adult↗
PubMed1996

Influence of native and modified lipoproteins on migration of mouse peritoneal macrophages and the effect of the antioxidants vitamin E and Probucol.

Macrophage-derived foam cells are a key component of the atherosclerotic plaque. Stimulation of the migration of foam cells back to the blood potentially offers an approach to plaque regression. However, information on the natural and synthetic factors influencing macrophage migration remains limited. The aim of the present study was to determine the effects of different lipoproteins and antioxidants on the migration of mouse peritoneal macrophages and macrophage-derived foam cells. The migration of cells was determined in vitro by using a Boyden chamber. Cellular content of cholesterol and cholesteryl esters was measured by HPLC. The native lipoproteins, VLDL, LDL, and HDL, and the chemically modified lipoproteins acetylated LDL (acLDL) and oxidized LDL (oxLDL), all acted as chemoattractants for macrophages that had not been lipid-loaded. The antioxidants vitamin E and Probucol had chemoattractive and chemokinetic effects on the migration of unloaded mouse peritoneal macrophages. Foam cells (lipid-laden macrophages) showed decreased mobility with increased cellular lipid content. However, as macrophages accumulated higher amounts of cholesterol and cholesteryl esters, the additional decrease of cell migration was relatively minor. Simultaneous incubation of macrophages with acLDL and vitamin E or Probucol increased migration. The content of cellular cholesterol and cholesteryl esters was not changed when acLDL-treated cells were exposed at the same time to vitamin E, but cholesteryl ester content was increased in the presence of Probucol. Overall, the findings indicate that the antioxidants vitamin E and Probucol have a potential anti-atherogenic action in stimulating migration of lipid-laden macrophages without major impairment of the ability of the cells to accumulate and metabolize modified LDL.

Animals↗
PubMed1995

Microscopy of the gap junction: a historical perspective.

Gap junctions were discovered more than three decades ago, and since this time, enormous strides have been made in understanding their structure and function. This article summarises the part played by microscopy, within the context of multidisciplinary research, in the historical development of our knowledge of the gap junction.

Animals↗
PubMed1995

Association of granulocyte-macrophage colony-stimulating factor with the crystalloid granules of human eosinophils.

We have previously shown that normal-density human peripheral blood eosinophils transcribe and translate mRNA for granulocyte-macrophage colony-stimulating factor (GM-CSF) and that the intracellular distribution was granular as assessed by light microscopy immunocytochemistry. The present study was conducted to confirm this apparent association between GM-CSF and the crystalloid granule using a subcellular fractionation method for human eosinophils and immunogold electron microscopy (EM). Highly purified (> 99%, by negative selection using anti-CD16 immunomagnetic microbeads) human peripheral blood eosinophils were obtained from four asthmatic subjects (not taking systemic medication), homogenized and density fractionated (5 x 10(7) cells/subject) on linear Nycodenz gradients. Twenty-four fractions were collected from each cell preparation and analyzed for marker enzyme activities as well as total protein. Dot blot analysis with specific monoclonal antibodies (MoAbs) was used to detect the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP). An anti-CD9 MoAb was used as an eosinophil plasma membrane marker. Lactate dehydrogenase (LDH) was used as a cytosolic marker. Immunoreactivity for GM-CSF was detected by a specific enzyme-linked immunosorbent assay using a polyclonal antihuman GM-CSF antibody and confirmed by dot blot. GM-CSF coeluted with the cellular fractions containing granule markers (MBP, ECP, eosinophil peroxidase, hexosaminidase, and arylsulphatase), but not those containing cytoplasm (LDH+) or membrane (CD9+) markers. EM examination of pooled fractions associated with the peak of GM-CSF immunoreactivity confirmed that they contained crystalloid and small granules, but not plasma membrane. In addition, quantification, using immunogold labeling with an anti/GM-CSF MoAb, indicated preferential localization of gold particles over the eosinophil granule cores of intact cells. Thus, our results indicate that GM-CSF resides as a granule-associated, stored mediator in unstimulated human eosinophils.

Asthma↗
PubMed1995

Repression of the macrophage scavenger receptor in macrophage-smooth muscle cell heterokaryons.

Macrophage scavenger receptors mediate the uptake of chemically modified LDL in an unregulated manner, leading to massive intracellular accumulation of lipid and thus a foamy cellular morphology. In atherosclerotic lesions, foam cells originate not only from macrophages but also from smooth muscle cells, yet smooth muscle cells do not normally express scavenger receptors, and when exposed to chemically modified LDL in vitro, lipid accumulation does not occur. The mechanism of conversion of smooth muscle cells into foam cells in the arterial wall is thus still under discussion. To investigate whether direct interaction between macrophages and smooth muscle cells may be involved and to explore the effects of components of the two cell types on the expression of scavenger receptors, we report here experiments using somatic cell hybrids formed by fusion of the two cell types. Immunofluorescent labeling and confocal microscopic techniques were applied to investigate and measure (1) lipid accumulation (using Nile Red staining), (2) the binding and uptake of acetylated LDL (using 1,1'-dioctadecyl-1-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-labeled acetylated LDL), and (3) receptor expression (assessed using a specific anti-receptor antibody) in smooth muscle cell-macrophage heterokaryons, macrophage-macrophage homokaryons, smooth muscle cell-smooth muscle cell homokaryons, and unfused macrophages and smooth muscle cells. The results demonstrate that scavenger receptor expression becomes repressed in macrophage-smooth muscle cell heterokaryons but not in macrophage-macrophage homokaryons. One possible explanation for the observed repression would be the existence of a negative regulatory cytoplasmic factor produced by smooth muscle cells.

Animals↗
PubMed1995

Upregulation of connexin43 gap junctions during early stages of human coronary atherosclerosis.

Interactions between cells form the framework for understanding the pathogenesis of atherosclerosis, but little information is available on the role of direct intercellular communication via gap junctions in this process. To investigate gap junction expression in the pathogenesis of human atherosclerosis, lesions representing different stages of the disease were obtained from coronary arteries of hearts removed from patients undergoing cardiac transplantation. Twelve hearts, each providing 1 to 3 segments of artery, were used in the study. Sections were examined by confocal laser scanning microscopy after immunofluorescent labeling with a specific antibody against connexin43, the major gap-junctional protein of smooth muscle cells, to permit high-definition visualization of immunolabeled gap junctions through the depth of the specimen. Double labeling using anti-connexin43 and cell type-specific antibodies demonstrated colocalization of gap junctions with smooth muscle cells but not with macrophages, a relationship confirmed by electron microscopy. Regions of intimal thickening and early atheromatous lesions showed markedly increased expression of connexin43 gap junctions between intimal smooth muscle cells compared with the undiseased vessels. This increase in gap junctions was most marked in regions of intimal thickening, semiquantitative analysis of the confocal digital images revealing a > 10-fold increase compared with the undiseased vessel. The quantity of labeled gap junctions in early atheromatous lesions, although higher than that of the undiseased vessel, was lower than that of intimal thickenings, and this trend toward reduced levels of gap junction immunolabeling with lesion progression continued, the value observed in the most advanced atheromatous lesions being lower than that of the undiseased vessel. As the quantity of gap junctions declined, their distribution became more patchy and the sizes of individual junctions larger. The results suggest that enhanced expression of gap junctions between smooth muscle cells may play a role in maintaining the synthetic phenotype during early growth of the atherosclerotic plaque.

Aged↗
PubMed1995

Cardiac muscle cell interaction: from microanatomy to the molecular make-up of the gap junction.

Electrical coupling between cardiac muscle cells is mediated by specialised sites of plasma membrane interaction termed gap junctions, one of three types of intercellular junction of the cardiac intercalated disk. Gap junctions consist of clusters of plasma membrane channels directly linking the cytoplasmic compartments of neighbouring cells. Gap-junctional channels are constructed from connexins, a multigene family of conserved proteins. The principal connexin isoform of the mammalian heart is connexin43; other connexins, notably connexin40, connexin45 and connexin37, are also expressed but in smaller quantities. Antibodies directed against unique sequences of these molecules allow investigation of the role of gap junctions and their component connexins in relation to the electrophysiological properties of the healthy and diseased heart. Confocal laser scanning microscopy of working ventricular myocytes immunolabelled with anticonnexin43 antibodies permits highly sensitive detection of gap junctions, allowing detailed analysis of the spatial distribution of the conduction pathways from the level of the cell to that of the tissue as a whole. Gap junction distribution, number and regional variations in the type of connexin expressed all contribute to the uniform anisotropic pattern of impulse spread characteristic of normal myocardium and the orderly, sequential contraction of the cardiac chambers. Connexin40 is preferentially expressed by myocytes of the atrioventricular conduction system and represents a specialisation facilitating fast conduction, allowing rapid distribution of the impulse throughout the working ventricle. Two major abnormalities in connexin43 gap junctions are detected in human ischaemic heart disease. First, at border zones adjacent to infarct scars, zones which are particularly prone to re-entry arrhythmia, there is marked disruption of the usual ordered distribution pattern of gap junctions. Second, a widespread downregulation of connexin43 gap junctions occurs in myocardium distant from the infarct, a change that is also found in the hypertrophic (non-ischaemic) heart. Consequent localised heterogeneous conduction and reduced conduction velocity provide an explanation for the genesis of re-entry arrhythmias. A current working hypothesis is that reduction in connexin43 gap junctions is a general pathogenetic feature of cardiac disease which predisposes the heart to arrhythmia, and that this reduction may form part of a wider pattern of alteration in the levels of other connexin isoforms.

Animals↗
PubMed1994

Gap junction alterations in the failing heart.

Electrical and mechanical integration between myocytes is mediated by three types of intercellular junction, the fascia adherens, desmosome and gap junction. Gap junctions are responsible for electrical coupling, and consist of clusters of plasma membrane channels that directly link the cytoplasmic compartments of neighbouring cells. Each channel consists of two hemichannels (connexons; one from each plasma membrane) aligned across the narrow extracellular gap, and each hemichannel is constructed from six connexin molecules. Using specific anticonnexin43 antibodies for immunofluorescence localization in combination with confocal laser scanning microscopy, alterations in the expression of connexin43 gap junctions have been investigated in chronic ischaemic heart disease and heart failure due to ischaemic cardiomyopathy. Two major alterations are apparent: (1) disturbance in the spatial distribution of gap junctions at the border zone of healed infarcts, and (2) reduction in the quantity of immunodetectable connexin43 in regions of normal gap junction distribution distant from infarct scars. These changes are likely to contribute to electromechanical dysfunction in ischaemic heart disease and heart failure, and appear to form part of a wider pattern of altered expression of different connexin types in the diseased heart.

Connexin 43↗
PubMed1994

Ultrastructural analysis of skeletal muscle. Microvascular dimensions and basement membrane thickness in chronic heart failure.

Chronic heart failure (CHF) is characterized by increased systemic vascular resistance and diminished blood flow to exercising skeletal muscle. The pathogenesis of the increased resistance is not known, and may be due to muscle atrophy, functional abnormalities of resistance vessels or to structural changes in the microcirculation such as endothelial cell swelling. We have compared the ultrastructure of the microvasculature in needle biopsies of the quadriceps muscle from seven control subjects with normal left ventricular function to 10 patients with moderate or severe heart failure, optimally treated and without evidence of fluid overload. Samples were processed for ultrathin sectioning using ruthenium red as a specific basement membrane (BM) stain. Electron micrographs were taken of 10 transversely cut capillaries from each specimen. The total cross-sectional area of the vessels and the area of the endothelium was determined, and the short axis diameter was measured as an index of vessel diameter. The BM thickness was calculated from the mean of six readings around the periphery of the vessel. The short axis diameter in the two groups was not significantly different (controls 3.37 +/- 0.21 microns, CHF 3.56 +/- 0.37 microns, mean +/- 1SD). No difference in total cross-sectional area (controls 11.64 +/- 1.86 microns 2, CHF 13.56 +/- 2.78 microns 2) or area of the endothelium (controls 4.90 +/- 1.18 microns 2, CHF 6.00 +/- 1.58 microns 2) was observed. The thickness of the BM was marginally increased in subjects with CHF when compared to control subjects (0.31 +/- 0.077 microns vs 0.246 +/- 0.047 microns, P = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

The Wolff-Parkinson-White syndrome: the cellular substrate for conduction in the accessory atrioventricular pathway.

The longstanding quest for the anatomical basis of the Wolff-Parkinson-White syndrome has left many unanswered questions. The ultrastructural morphology of the myocytes comprising accessory atrioventricular pathways, which are capable of rapid and variable conduction, is central to understanding the development and behaviour of this congenital anomaly, but remains unknown. Examination of three surgically resected pathways was performed to determine their underlying cellular morphology and the pattern of intercellular coupling, by correlative light microscopy, electron microscopy and confocal scanning laser microscopy combined with immunohistochemical localization of the cardiac gap-junctional protein, connexin43. Two left-sided pathways were composed of myocardium of 'normal working ventricular' type. The right-sided pathway was composed almost entirely of highly abnormal myocytes characterized by aberrant myofibril organisation, with a lack of A-band material and abnormal mitochondria, but normal intact intercalated disks no different from those seen in left-sided pathways. The gap junctions of all pathways were composed of connexin43 distributed as in ventricular myocardium, and not as found in atrial or atrioventricular nodal tissues. While myocytes of abnormal structure were present in one of the accessory atrioventricular pathways examined, all pathways had morphologically normal gap junctions, the structures responsible for efficient intercellular coupling, with a pattern of distribution suggestive of working ventricular myocardium.

Adolescent↗
PubMed1994

Pathophysiology of gap junctions in heart disease.

Electrical coupling between cardiac muscle cells is mediated by specialized sites of plasma membrane interaction termed gap junctions. These junctions consist of clusters of membrane channels that directly link the cytoplasmic compartments of neighboring cells. Each gap-junctional channel consists of two connexons, one from each of the interacting plasma membranes, extending across the narrow extracellular gap. Connexons are constructed from connexins, a multigene family of conserved proteins. Different connexins confer specific electrophysiologic characteristics on the assembled channel protein. The major connexin of the mammalian heart is connexin43, although other types of connexins are also expressed, notably connexin40 in myocytes of the atrioventricular conduction system. Confocal laser scanning microscopy of anti-connexin43 immunolabeled samples reveals two major abnormalities in myocardial gap junctions in ischemic heart disease: loss of the usual ordered distribution of gap junctions at border zones adjacent to infarct scars, and reduction in the quantity of connexin43 gap junctions in myocardium distant from the infarct. These and other changes reported in myocardial gap-junctional communication pathways following infarction may result in heterogeneous anisotropic conduction and reduced conduction velocity, thereby forming a proarrhythmic substrate. Current evidence suggests that reduction in connexin43 content is a general pathogenetic feature of cardiac disease, and that changes in the expression levels of other connexin types may contribute to altered electrophysiologic function in the diseased heart.

Animals↗
PubMed1994

Spatiotemporal relation between gap junctions and fascia adherens junctions during postnatal development of human ventricular myocardium.

BACKGROUND: The growing postnatal human heart maintains electromechanical function while undergoing substantial changes of cellular topology and myocardial architecture. The capacity for growth and remodeling of ventricular myocardium in adaptation to the hemodynamic changes of early infancy later declines. This decline is associated with changes in electromechanical properties of the myocardium, which suggest that the electrical and mechanical interactions between the myocytes may change in an age-dependent manner. Thus, reduction in the capacity for myocardial growth and adaptability may relate to age-dependent alterations in the patterns of the intercellular junctions that mediate electrical and mechanical coupling. We therefore examined the hypotheses that (1) age-dependent changes in the distribution patterns of gap junctions and fasciae adherentes, the intercellular junctions responsible, respectively, for electrical and mechanical coupling, accompany postnatal development in the human heart and that (2) such changes continue into the first few years of childhood. Further, the spatial relation between the two types of junction, for which a close association has been hypothesized as necessary, was explored. METHODS AND RESULTS: Ventricular myocardial gap-junction distribution was investigated in 23 pediatric surgical patients (4 weeks to 15 years old) by quantitative immunohistochemical localization of the principal cardiac gap-junctional protein, connexin43, using confocal microscopy. Immunolocalization of fascia adherens junctions by labeling N-cadherin, and correlative immunogold and standard electron microscopy, were performed in parallel. In the neonate, connexin43 gap junctions have a punctate distribution over the entire surface of the ventricular myocytes. With advancing age, gap junctions become progressively confined to the transverse terminals of the cell, ie, toward the distribution within the intercalated disk characteristic of the adult ventricle. The transversely arrayed proportion of gap-junctional label showed a linear increase with age (R = .88, P < .001), reaching the adult pattern at about 6 years, and the fascia adherens junctions showed a similar progression. Electron microscopy confirmed the changing pattern of junctional contacts and demonstrated that initially gap junctions and adhering junctions are frequently not closely adjacent but become increasingly so with maturation of the intercalated disk. CONCLUSIONS: Changes in the spatiotemporal patterns of the intercellular junctions responsible for electrical and mechanical coupling are closely coordinated in postnatal human ventricular myocardium and continue to about 6 years of age. Over this period there is a close and increasing association between the gap junctions and fascia adherens junctions. These changes in the distribution of intercellular electrical and adhering junctions may parallel the changing functional requirements of the ventricle, from a distribution that facilitates the remodeling necessitated by rapid growth and changing hemodynamics to that of the relatively stable and rapidly conducting adult myocardium. These age-related changes may also diminish the ability for appropriate myocardial remodeling in response to physiological, pathological, or surgical hemodynamic alterations.

Adolescent↗
PubMed1994

A comparison between the epithelial tight junction morphology of human extrapulmonary bronchi and rat trachea.

Animal models have been used to investigate the involvement of epithelial tight junctions in the pathogenesis of human airway disease. However, no previous study has compared the tight junction morphologies of human and animal species in order to relate findings in animal models to human disease. In the present study, we therefore undertook a comprehensive quantitative evaluation of tight junction morphology, to determine what similarities or differences may exist in rat and human airways. Human tissue was obtained from grossly and histologically normal extrapulmonary bronchi from lungs resected for pulmonary tumour (n = 8); rat tracheal epithelium was acquired from Sprague-Dawley specific pathogen-free animals (n = 12). The tight junction morphologies of the two species were compared with respect to junctional depth, number of strands and junctional complexity. The basic architectural arrangement of the tight junctions in both species was found to be similar; however, tight junctions in rat tissues were less deep, comprised fewer strands, and had fewer strand interconnections compared with those in the human samples. The number of strands per interconnection was similar in the two species. We conclude that, in spite of a general similarity of rat and human airway epithelial tight junctions, there are specific quantitative details of morphology which need to be considered when attempting to extrapolate to the human the results of studies of airway epithelial permeability conducted in the rat. The precise biological significance of these differences, as yet, remains unclear.

Animals↗
PubMed1994

No effect of histamine on human bronchial epithelial cell permeability and tight junctional integrity in vitro.

Both animal and human studies have suggested that histamine increases airway epithelial cell permeability in vivo. In order to study the effect of histamine on paracellular epithelial permeability and tight junctional integrity, we have cultured human bronchial epithelial cells to confluency and investigated the effect of topically applied 0.1-20.0 microM histamine. Cultures were established on microporous membranes of tissue culture cell inserts and used for the assessment of: 1) transepithelial movement of radiolabelled mannitol (14C-mannitol) and bovine serum albumin (14C-BSA), in the luminal to serosal direction and 2) changes in electrical resistance of the cultures. Epithelial cell cultures were also established on plastic coverslips, in order to determine tight junction morphology by freeze-fracture electron microscopy, and to assess junctional integrity by lanthanum penetration, using thin sections. Compared with untreated control cultures, 0.1-10 microM histamine did not significantly alter the movement of either 14C-mannitol or 14C-BSA across the epithelial cultures at any time during incubation, but caused an increase in the electrical resistance of the cultures, which was maximal by 6 h of incubation. The morphology of the tight junctions revealed by freeze-fracture and junctional integrity (the latter determined by the degree of lanthanum penetration) were similar in untreated control cultures and cultures incubated with histamine. These studies indicate that histamine does not have a direct effect on paracellular bronchial epithelial permeability in vitro.

Animals↗
PubMed1993

Stretch and anesthetic dependency of atrial natriuretic peptide release demonstrated by an ultrastructural assay.

Using an ultrastructural assay developed to quantify the secretion of atrial natriuretic peptide-containing granules, release of the hormone, in response to different degrees of atrial distension, is directly demonstrated at the cellular level. The ultrastructural assay developed uses an in situ tannic acid perfusion technique to arrest the exocytosis of atrial granules in the anesthetized rat. Secretory granules, which retain the capacity to undergo exocytosis throughout a 30-minute tannic acid perfusion, accumulate at the cell surface in a state of fusion with the plasma membrane, with the core contents retained. Quantification of arrested granules thus provides a measure of the rate of granule release and allows the responses to different stimuli to be assessed. By altering the height of the perfusate, perfusion pressure and hence the degree of distension of the right atrium can be increased, and this causes a proportional rise in the release of secretory granules from individual myocytes. An anesthetic regime incorporating fentanyl citrate was found to increase significantly the rate of granule release, and this was further augmented by atrial distension. Quantification of the numbers of cytoplasmic granules under the same conditions did not reveal a reduction in granules. This is thought to be because only a small pool of granules is recruited for exocytosis, and granule production may continue during the perfusion period. Our assay of atrial secretory granule release allows the effect of a variety of stimulatory and inhibitory agents to be assessed directly at the cellular level and provides an independent comparison with previous biochemical data from whole animal and isolated organ studies.

Anesthetics↗
PubMed1993

Expression of connexin43 gap junctions between cultured vascular smooth muscle cells is dependent upon phenotype.

The smooth muscle cell is the predominant cell type of the arterial media. In the adult vascular system, smooth muscle cells are found primarily in the contractile phenotype, but following injury or during atherosclerotic plaque formation the secretory synthetic phenotype is expressed. Recently it has been shown that gap junction connexin43 messenger RNA levels are six times higher in cultured smooth muscle cells in the synthetic phenotype than in intact aorta. We have modulated rabbit aortic smooth muscle cells in culture between the synthetic phenotype and one resembling the contractile phenotype, and correlated gap junction expression with phenotype. A dual labelling technique with antibodies against smooth muscle myosin and a synthetic peptide constructed to match a portion of the connexin43 gap junction protein was used for these experiments. Gap junctions are numerous between synthetic phenotype cells but few are observed between contractile cells. Rat aortic smooth muscle cells were also cultured and the growth and structure of gap junctions followed in the synthetic phenotype by use of freeze-fracture electron microscopy and immunohistochemical techniques. Junctional plaques are similar in structure to those observed in cardiac muscle, their size and number increasing with time in culture. The increased numbers of gap junctions between synthetic phenotype smooth muscle cells may be important during vessel development, following injury, or in atherosclerotic plaque formation.

Animals↗