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N J Severs

Publications and source records attributed to N J Severs.

At least 19 recordsLinked to original sources
PubMed1997

Disturbed connexin43 gap junction distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia.

BACKGROUND: Slow, nonuniform conduction caused by abnormal gap-junctional coupling of infarct-related myocardium is thought to be a component of the arrhythmogenic substrate. The hypothesis that changes in gap-junctional distribution in the epicardial border zone (EBZ) of healing canine infarcts define the locations of reentrant ventricular tachycardia (VT) circuits was tested by correlating activation maps of the surviving subepicardial myocardial layer with immunolocalization of the principal gap-junctional protein, connexin43 (Cx43). METHODS AND RESULTS: The EBZ overlying 4-day-old anterior infarcts in three dogs with inducible VT and three noninducible dogs was mapped with a high-resolution electrode array and systematically examined by standard histology and confocal immunolocalization of Cx43. The thickness of the EBZ was significantly less in the hearts with (538 +/- 257 microns) than without (840 +/- 132 microns; P < .05) VT. At the interface with the underlying necrotic cells, the EBZ myocardium showed a marked disruption of gap-junctional distribution, with Cx43 labeling abnormally arrayed longitudinally along the lateral surfaces of the cells. In the EBZ of all hearts, the disrupted Cx43 labeling extended part of the way to the epicardial surface, with the most superficial epicardial myocytes having the normal transversely orientated pattern. Only in the hearts with inducible VT did the disorganization extend through the full thickness of the surviving layer at sites correlating with the location of the central common pathways of the figure-of-8 reentrant VT circuits. CONCLUSIONS: Altered gap-junctional distribution is part of the early remodeling of myocardium after infarction, and by defining the location of the common central pathway of the reentrant VT circuits, it may be a determinant of VT susceptibility.

Animals↗
PubMed1997

Connexin43 gap junction levels during development of the thoracic aorta are temporally correlated with elastic laminae deposition and increased blood pressure.

A characteristic property of the vascular smooth muscle cell is its ability to modulate between a contractile phenotype, responsible for control of vascular tone, through to a synthetic phenotype, capable of migration and synthesis of extracellular matrix molecules. Smooth muscle cells are coupled by gap junctions, the membrane structures which permit direct intercelluar passage of ions and small molecules, and which play a role both in electrical coupling and intercellular communication during patterning and development. We have previously found that connexin43 type gap junction expression is upregulated in the synthetic phenotype smooth muscle cell in vitro and during atherosclerotic plaque formation in human coronary arteries. On the basis of immunohistochemical labelling, confocal laser scanning microscopy and digital image analysis, we now report that relatively high levels of connexin43 are expressed during development of the rat thoracic aorta, temporally correlating with reported periods of smooth muscle cell proliferation and secretion of elastic laminae. A major peak in expression occurs at seven days post-natal, with a second less pronounced peak at 72 days post-natal. The principal peak in gap junction levels appears to coincide with increased post-natal blood pressure and aorta media thickening. The amount of gap junction labelling falls off to normal adult levels as the smooth muscle cells modulate towards the contractile phenotype and growth is completed. The results indicate an association between direct cell-to-cell communication and synthetic phenotype smooth muscle cell activity during aortic growth and patterning.

Animals↗
PubMed1997

Angiotensin II receptor type 1 mRNA is upregulated in atria of patients with end-stage heart failure.

There is increasing evidence that pathological changes in the myocardium during chronic heart failure (CHF) are partly regulated through the activation of the renin-angiotensin system (RAS), an effect mediated by the angiotensin II type 1 receptor (AT1R). We examined the expression of cardiac AT1R mRNA in normal (atria, n=7; ventricle, n=3) and end-stage CHF human hearts (atria, n=8; ventricle, n=14). Tissue was snap-frozen immediately after explantation during orthotopic cardiac transplantation; control specimens were obtained from healthy donor hearts rejected for technical reasons. Northern blots of purified total mRNA from each tissue were hybridized with a random primed radiolabeled probe for the coding sequence of AT1R. Stringent conditions were used for both hybridization (5X SSC, 65 degrees C) and washing (0.5X SSC, 0.1% SDS, 65 degrees C) of the membrane. Left and right atrial tissue showed low levels of AT1R mRNA expression in the controls, with statistically significant upregulation of expression in tissue from pathological hearts; CHF atria 1.28+/-0.86 optical density (OD) units, control atria 0.56+/-0.31 OD units, P=0.05 (mean+/-s.d.). There were undetectable levels in ventricles from either control (2/2) or dilated hearts (7/7). The results were independent of the etiology of the heart failure and suggest that increased levels of atrial AT1R mRNA may occur in response to elevated atrial pressures in heart failure.

Adult↗
PubMed1997

Quantitative immunoconfocal analysis of human myometrial gap junction connexin43 in relation to steroid hormone concentrations at term labour.

The aim of this study was to quantify gap junction expression in the human myometrium in relation to progesterone and oestradiol concentrations, and to establish whether oxytocin-resistant dystocia is due to an abnormality in gap junction expression. Three groups of patients were investigated: (i) before labour (at term), (ii) normal labour and (iii) oxytocin-resistant dystocia (eight patients per group). For each patient, the concentrations of oestradiol and progesterone in maternal blood and in myometrial tissue were measured, and the number and area of immunostained connexin43 gap junctions per unit volume of tissue determined by quantitative analysis of digital images obtained by confocal microscopy. No significant difference in connexin43 gap junction content was observed between the three patient groups. When all groups were pooled, there was a significant positive correlation (P < 0.05) between the quantity of immunolabelled gap junctions and the oestradiol:progesterone ratio, but there was no significant difference in this correlation between the groups. Gap junction immunolabelling was not correlated with the progesterone or oestradiol concentration in the maternal blood or the myometrium. These data suggest that in human myometrium: (i) dystocia is not due to a reduced level of immunodetectable connexin43 gap junctions, (ii) onset of labour is not associated with a sudden increase in immunodetectable gap junction protein and (iii) gap junctions can be expressed in the presence of high progesterone concentrations.

Adult↗
PubMed1997

Spatial distribution of dihydropyridine receptors in the plasma membrane of guinea pig cardiac myocytes investigated by correlative confocal microscopy and label-fracture electron microscopy.

Excitation-contraction coupling in cardiac muscle is thought to depend fundamentally on the spatial organization of sarcolemmal dihydropyridine receptors (L-type calcium channels) in relation to ryanodine receptors (calcium-release channels of the sarcoplasmic reticulum). In the present study, we have investigated the distribution of dihydropyridine receptors in the guinea pig myocyte plasma membrane by correlative immunoconfocal microscopy and label-fracture electron microscopy. Label-fracture, a method in freeze-fracture cytochemistry, permits immunogold localization of cell surface proteins in en face membrane views. Taken together, results from confocal microscopy and label-fracture replicas suggest that, in the peripheral plasma membrane, calcium channels are organized predominantly in the form of clusters. Confocal microscopy also suggests a similar organization in the transverse tubules. It is hypothesized that these clusters may lie adjacent to junctional sarcoplasmic reticulum, permitting the close coupling of influx of calcium through plasma membrane calcium channels to trigger release of calcium from the intracellular stores, as part of the mechanism of calcium-induced calcium release.

Animals↗
PubMed1997

Dissociated spatial patterning of gap junctions and cell adhesion junctions during postnatal differentiation of ventricular myocardium.

Nonuniformity in the spatial patterning of gap junctions between heart muscle cells is now recognized as an important determinant of electromechanical function in working myocardium. Breakdown of the normal geometry of electrical intercellular connectivity in diseased myocardium correlates with reentry, arrhythmia, and conduction disturbance. The developmental mechanism(s) that determines this precise spatial order in gap junction organization in normal myocardium is at present unknown. To examine this question, we have used immunoelectron and immunoconfocal microscopy to analyze the spatial distributions of gap junctional (connexin43), desmosomal (desmoplakin), and adherens junctional (N-cadherin) components during maturation of rodent and canine left ventricular myocardium. In rats, a striking divergence in the distribution of gap junctions and cell adhesion junctions emerged within the first 20 days of postnatal life. It was found that although gap junctions initially demonstrated dispersed distributions across myocyte cell membranes, desmosomes and adherens junctions showed more rapid polarization toward cell termini (ie, nascent intercalated disks) after birth. Over subsequent postnatal development (20 to 90 postnatal days), gap junctions became progressively concentrated in these cell adhesion junction-rich zones of membrane. Quantitative analyses of this process in a series of rats aged 15 embryonic and 1, 5, 10, 20, 40, 70, and 90 postnatal days indicated that significantly higher levels (P < .01) of N-cadherin and desmoplakin than of connexin43 were immunolocalized to cell termini by as early as postnatal day 5. Although all three junctions types showed increasing polarization to myocyte termini with development, variation between junctions remained significant (P < .05) at all times points between 5 and 70 postnatal days. Only at 90 postnatal days, when the animals were nearly full grown, did the proportions of gap junction, desmosome, and adherens junction at intercalated disks become statistically similar (P > .05). Examination of myocardium from 1- and 3-month-old canines revealed that related differential changes to the spatiotemporal distribution of intercellular junctions occurred during postnatal maturation of the dog heart, suggesting that the process was not rodent specific. It is concluded that this progressive change in the organization and pattern of association between gap junctions and cell adhesion junctions is likely to be an important factor in maturation of electromechanical function within the mammalian heart.

Animals↗
PubMed1997

Dystrophin is not a specific component of the cardiac costamere.

Dystrophin is a key component of the subsarcolemmal skeleton of muscle cells, and lack of dystrophin is the direct cause of Duchenne muscular dystrophy. In skeletal muscle, dystrophin is reported to be localized specifically at costameres, transversely oriented riblike subsarcolemmal plaques that mechanically couple the contractile apparatus to the extracellular matrix. Costameres are characteristically rich in vinculin and are prominent in cardiac as well as skeletal muscle. To define the precise spatial relationship between dystrophin in relation to the costamere in cardiac muscle, we applied high-resolution single- and double-immunolabeling techniques, under a range of preparative conditions, with visualization of vinculin (as a costamere marker) and dystrophin by confocal microscopy and by the freeze-fracture cytochemical technique, fracture label. Immunoconfocal visualization revealed dystrophin as a continuous uniform layer at the cytoplasmic surface of the peripheral plasma membrane of the rat cardiac myocyte at both costameric and noncostameric regions. The pattern of labeling was reproducible with three different antibodies and was independent of time and antibody concentration. Platinum/carbon replicas and thin sections of fracture-label specimens permitted high-resolution visualization of the distribution of dystrophin in plane views of the freeze-fractured plasma membrane and in relation to the sarcomeric banding patterns of the underlying myofibrils. These results confirmed no preferential association of dystrophin with costameres or with any region of the sarcomeres of underlying myofibrils in rat cardiac tissue. We conclude that in contrast to skeletal muscle, dystrophin in cardiac muscle is not exclusively a component of the costamere.

Animals↗
PubMed1997

Gap junction localization and connexin expression in cytochemically identified endothelial cells of arterial tissue.

Vascular endothelial cells interact with one another via gap junctions, but information on the precise connexin make-up of endothelial gap junctions in intact arterial tissue is limited. One factor contributing to this lack of information is that standard immunocytochemical methodologies applied to arterial sections do not readily permit unequivocal localization of connexin immunolabeling to endothelium. Here we introduce a method for multiple labeling with specific endothelial cell markers and one or more connexin-specific antibodies which overcomes this limitation. Applying this method to localize connexins 43, 40, and 37 by confocal microscopy, we show that the three connexin types have quite distinctive labeling patterns in different vessels. Whereas endothelial cells of rat aorta and coronary artery characteristically show extensive, prominent connexin40, and heterogeneous scattered connexin37, the former, unlike the latter, also has abundant connexin43. The relative lack of connexin43 in coronary artery endothelium was confirmed in both rat and human using three alternative antibodies. In the aorta, connexins43 and 40 commonly co-localize to the same junctional plaque. Even within a given type of endothelium, zonal variation in connexin expression was apparent. In rat endocardium, a zone just below the mitral valve region is marked by expression of greater quantities of connexin43 than surrounding areas. These results are consistent with the idea that differential expression of connexins may contribute to modulation of endothelial gap junction function in different segments and subzones of the arterial system.

Animals↗
PubMed1996

Effect of neuromimetics upon the release of atrial natriuretic peptide granules: are multiple pathways involved in secretion?

The release of atrial natriuretic peptide (ANP) in response to the application of neurohumoral agonists (neuromimetics) is directly demonstrated and quantified at the cellular level, using an ultrastructural assay developed to quantify secretion. The assay uses an in situ tannic acid perfusion technique to arrest the exocytosis of atrial secretory granules in the anesthetized rat. The animal is perfused with the neuromimetic, and secretory granules, which retain the capacity to undergo exocytosis throughout the subsequent 30 min tannic acid perfusion, accumulate at the cell surface in a state of fusion with the plasma membrane. Quantification of arrested granules thus provides a measure of the rate of granule release and allows the responses to different agents to be assessed. The actions of three different agents were investigated: isoproterenol, phenylephrine, and acetylcholine. In previously published studies, investigations of the actions of these agents on ANP release has produced unclear and sometimes contradictory results. Using our ultrastructural assay, it was found that during the 30 min perfusion period neither isoprenaline nor phenylephrine caused a significant change in the rate of secretory granule release, whereas acetylcholine significantly decreased the rate of granule release. A new model of secretion is proposed to integrate these findings with previous results and help clarify the complex picture of atrial natriuretic peptide release.

Acetylcholine↗
PubMed1996

Effects of calcium on the migration and recruitment of macrophages and macrophage-derived foam cells.

Calcium is thought to play an important role in the genesis of atherosclerotic lesions, but the precise mechanisms involved are unclear. In the present investigation, we have used in vitro systems to investigate the effects of calcium on one key aspect of lesion development: the migration of macrophages and macrophage/foam cells. Using agarose plate migration assays, the migratory characteristics of macrophages exposed to 1) no lipoprotein, 2) low density lipoprotein (LDL), 3) acetylated low density lipoprotein (acLDL), and ) oxidized low density lipoprotein were examined. The most marked stimulatory effect on macrophage mobility was observed when freshly isolated cells were exposed to acLDL during the migration assay. High levels of exogenous calcium were found to suppress the stimulatory effect of acLDL on migration. As the responses of macrophages exposed to a uniform concentration of agents in the surrounding medium may differ from chemotactic responses to a concentration gradient, the migration of macrophages, with and without preexposure to acLDL or LDL, was studied using microchemotaxis Boyden chambers. Under these conditions, calcium acted as a highly potent chemoattractant, especially to cells that had been preincubated with acLDL. These results suggest how elevated external calcium concentration leads initially to macrophage recruitment, and subsequently to foam cell aggregation and lipid core formation, in association with calcification, in the developing atherosclerotic plaque.

Animals↗
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↗