Smoothelin in adult and developing human arteries and myocardium

The aim of this investigation was to study, with immunohistochemical methods, the distribution of the novel cytoskeletal protein smoothelin in human cardiovascular tissues, the possible changes during the development of the cardiovascular system and its correlation to the intermediate filament proteins desmin and vimentin. Smoothelin was detected in smooth muscle cells of the fetal coronary arteries. In very young subjects (up to 3 months of age), only a few cells in the media of the elastic arteries contained smoothelin, whereas it was present in most smooth muscle cells in the muscular arteries. In individuals older than 1 year, most smooth muscle cells in the media of all blood vessels contained smoothelin. In vessels with a developed intima, smoothelin was present in a variable proportion of the smooth muscle cells. With few exceptions, smoothelin was more frequently detected than desmin in medial smooth muscle cells. Smoothelin and vimentin were codistributed in the smooth muscle cells of the media in most vessels. In the cardiomyocytes (fetal to adult age), the smoothelin antibody detected epitopes located at the Z-disc level but not in the intercalated discs. In conclusion, smoothelin is more widely distributed in the muscular arteries than in the elastic arteries early in life, and thus exhibits a variable distribution during postnatal development of vascular tissues. In the adult, smoothelin is detected in the media of most vascular smooth muscle cells, both in muscular and elastic arteries, and is not necessarily codistributed with either desmin or vimentin. Evidence that smoothelin is present in human striated cardiomyocytes is also presented. Accepted: 16 July 1999     

Expression of intermediate filament-associated proteins paranemin and synemin in chicken development

The expression of two intermediate filament-associated proteins, paranemin (280,000 mol wt) and synemin (230,000 mol wt), was investigated with respect to the expression of two core intermediate filament proteins, desmin and vimentin, in various embryonic and adult chicken muscle and nonmuscle cells. All developing muscle cells, regardless of their type, simultaneously express desmin, vimentin, paranemin, and synemin. However, a difference is observed in the expression of paranemin in adult muscle. This protein is removed during differentiation of both fast and slow skeletal muscle, visceral smooth muscle, and the smooth muscle of muscular arteries, but remains in mature myocardial cells, cardiac conducting fibers, and the smooth muscle cells of elastic arteries. Some of these cells express vimentin, others desmin, and still others a mixture of the two. On the other hand, synemin is expressed in all the above types of adult muscle cells except myocardial cells. Adult myocardial cells also lack vimentin, and its presence is gradually reduced after hatching. Since in adult striated muscle all expressed intermediate filament proteins are found predominantly in association with the peripheries of myofibrillar Z discs, these results suggest that a change in the composition of skeletal and cardiac muscle Z discs occurs during chicken development and maturation. Erythrocytes that express synemin and vimentin do not express paranemin, while both embryonic and adult Schwann cells co- express paranemin and vimentin, but not synemin. Endothelial cells of muscular vessels express paranemin, while those of elastic vessels do not, and neither contains synemin. Paranemin and synemin are not expressed in neurons, epithelial, and most glial cells, suggesting that these two polypeptides are expressed only in conjunction with desmin or vimentin. These results suggest that the composition of intermediate filaments changes during chicken development, not only with respect to their core subunit proteins but also with respect to two associated polypeptides, particularly in muscle cells.     

Heterogeneity of Intermediate Filament Expression in Vascular Smooth Muscle: A Gradient in Desmin Positive Cells from the Rat Aortic Arch to the Level of the Arteria Iliaca Communis

The display of the two distinct intermediate filament proteins, desmin and vimentin, in rat vascular smooth muscle tissue was studied by immunofluorescence microscopy on frozen sections of aorta and other blood vessels. Vascular smooth muscle cells present in these vessels always appeared rich in vimentin. However, staining of sections covering six distinct but contiguous parts of the aorta showed that the number of desmin containing cells was low distal to the truncus brachiocephalicus, but increases until in distal parts of the aorta and in the arteria iliaca communis almost all cells appear positive for desmin. Thus blood vessels show heterogeneity of intermediate filament expression not only in cross-section but can also display heterogeneity along their length. Muscular arteries such as the renal artery and the arteria femoralis, as well as arterioles and veins including the vena jugularis and the vena cava also contain desmin. Thus it may be that low numbers of desmin-positive cells are typical of elastic arteries, while muscular arteries and other blood vessels are characterized by large numbers of desmin-positive cells. We discuss whether desmin-positive and desmin-negative vascular smooth muscle cells may perform different functions and raise the possibility that desmin expression may coincide with the turn on of a specially regulated contractility program.     

Effect of sympathectomy on the phenotype of smooth muscle cells of middle cerebral and ear arteries of hyperlipidaemic rabbits

This study was carried out to determine whether sympathectomy influences the phenotypic modulation of smooth muscle cells in the peripheral and cerebral arteries of heritable hyperlipidaemic rabbits. Unilateral superior cervical ganglionectomy (common origin of innervation to the middle cerebral artery and the central ear artery) was performed on four Watanabe heritable hyperlipidaemic rabbits. Cross-sections of the ipsi- (sympathectomized) and the contralateral (intact) cerebral and ear arteries were prepared 2 months later and labelled with monoclonal antibodies against vimentin and desmin, two markers of the differentiation of smooth muscle cells, and α-smooth muscle actin, a marker of these cells. Sections from control and sympathectomized arteries were analysed with a confocal laser scanning microscope. Compared with contralateral intact ear arteries, the sympathectomized ear artery developed a thickened intima with dedifferentiated smooth muscle cells, expressing α-smooth muscle actin but no desmin, whereas the middle cerebral artery remained unchanged. These results suggest that sympathectomy may favour the progression of atherosclerosis in peripheral but not in cerebral arteries of Watanabe heritable hyperlipidaemic rabbits     

Contractile and cytoskeletal proteins of smooth muscle cells in rat, rabbit, and human arteries

The aim of this study was to determine whether similar populations of smooth muscle cells, in relation to contractile and cytoskeletal proteins, are present in normal and diseased human coronary arteries and normal and injured rat and rabbit arteries. Rat aortae and rabbit carotid arteries were de-endothelialised and the resulting neointimal thickening examined at set time points 2-24 weeks later. Immunohistochemistry revealed that arteries had three distinct populations of cells in respect to alpha-smooth muscle actin, smooth muscle myosin heavy chain and vimentin (staining intensities '-', '+' or '++' for each protein), but only two populations in respect to desmin ('-' and '+'). The different populations of cells were found in the neointima at all times after injury, in human atherosclerotic plaque and in the media of diseased, injured and uninjured vessels, although in different proportions. It was concluded that arteries of the human, rat and rabbit have cells with a wide spectrum of contractile and cytoskeletal proteins. Expression of the different proteins did not reflect the state of the artery after injury or during the disease process, and was not associated with the expansion of a subset of cells within the artery wall.     

Heterogeneity of desmin,the muscle-type intermediate filament protein,in blood vessels and astrocytes

Summary Monoclonal antibodies were isolated from mice immunized with chicken gizzard desmin. Antibodies reacting with desmin on immunoblots and selectively decorating chicken and rat intestinal smooth muscle as well as the Z-line in striated muscle, were selected for this study. Based on their staining pattern on cryostat sections of chicken and rat cerebellum, spleen, kidney, aorta and femoral artery, monoclonal supernatants could be divided in three groups: (i) antibodies decorating astrocytes and vascular smooth muscle; (ii) antibodies decorating only vascular smooth muscle; (iii) antibodies decorating only astrocytes. Antibodies in group (i) and (iii) also stained GFA-negative Bergmann glia in chicken cerebellum. It is proposed that desmin may vary depending on the histological localization.     

Muscular ETB receptors develop postnatally and are differentially distributed in specific segments of the rat vasculature.

The endothelin/endothelin-receptor system is a key player in the regulation of vascular tone in mammals. We raised and characterized an antiserum against rat ETB receptor and investigated the distribution of ETB receptors in different vascular beds during postnatal development (day 0 through day 28) and in the adult rat. We report the tissue-specific and age-dependent presence of vasoconstrictor ETB receptors. At the time of birth, vascular smooth muscle cells from all tissues examined did not exhibit ETB receptor immunoreactivity. The occurrence of ETB receptor immunoreactivity in the postnatal development was time dependent and started in small coronary and meningeal arteries at day 5, followed by small mesenteric arteries as well as brachial artery and vein at day 14. At day 21, ETB receptors were present in the media of muscular segments of pulmonary artery, large coronary arteries, and intracerebral arterioles. At day 28, ETB receptor immunoreactivity was evident in interlobular renal arteries, vas afferens, and efferens. Large renal arteries, mesenteric artery, and elastic segments of pulmonary arteries, as well as coronary and mesenteric veins, did not exhibit ETB receptor immunoreactivity. These data demonstrate the age-dependent and tissue-specific presence of ETB receptors, mainly on arterial smooth muscle cells in the vascular system of the rat.     

Heterogeneity of desmin, the muscle-type intermediate filament protein, in blood vessels and astrocytes

Monoclonal antibodies were isolated from mice immunized with chicken gizzard desmin. Antibodies reacting with desmin on immunoblots and selectively decorating chicken and rat intestinal smooth muscle as well as the Z-line in striated muscle, were selected for this study. Based on their staining pattern on cryostat sections of chicken and rat cerebellum, spleen, kidney, aorta and femoral artery, monoclonal supernatants could be divided in three groups: (i) antibodies decorating astrocytes and vascular smooth muscle; (ii) antibodies decorating only vascular smooth muscle; (iii) antibodies decorating only astrocytes. Antibodies in group (i) and (iii) also stained GFA-negative Bergmann glia in chicken cerebellum. It is proposed that desmin may vary depending on the histological localization.     

The effect of prolonged ischaemia and cryopreservation on the cell viability of human aortic and femoral artery allografts

The viability of the human arterial allograft cells depends on the time and method of vessel procurement and storage. In this study, an evaluation of the effect of the duration of 4 degrees C ischaemia and cryopreservation on human aortic and femoral artery allograft viability was performed. After the isolation of arterial wall cells, the identification of cultured cells was performed using mRNA analysis for estimation of smooth-muscle markers of differentiation: desmin and heavy-caldesmon. The viability of cells from the medial layer of the aortic wall ranged from 74 to 90% (61-79% for femoral arteries). Cold ischaemia time (from harvesting until the beginning of the preparation) is a statistically significant factor influencing smooth muscle cell viability. Smooth muscle cells represented the majority of live cell population.     

Heparin inhibits proliferation of fetal vascular smooth muscle cells in the absence of platelet-derived growth factor

Proliferation of smooth muscle cells from the pulmonary arteries and aortas of fetal calves is inhibited by heparin in vitro. This effect is reversible and dose dependent. Comparisons with effects of other polysaccharides indicate that only extensively sulfated polysaccharides inhibit proliferation of smooth muscle cells but that specific structural features of heparin are required to achieve maximum effect. Heparin-Sepharose chromatography of medium containing fetal calf serum reduces the ability of that medium to promote growth of smooth muscle cells from fetal pulmonary arteries, suggesting that heparin may remove soluble growth factors in serum. However, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is identical in media supplemented either with serum prepared from fetal calf plasma, in which platelet-derived growth factor (PDGF) is not detectable, or with fetal calf serum, which contains relatively abundant PDGF (114 pg/ml). Thus, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is not mediated solely by decreased availability or activity of exogenous PDGF. These studies suggest that morphogenesis of the smooth muscle investment of the pulmonary arteries could be regulated by local production of heparin-like inhibitors of smooth muscle cell growth.     

Distribution of vimentin and desmin filaments in smooth muscle tissue of mammalian and avian aorta

The presence of intermediate filament proteins in vascular tissue cells has been examined by immunofluorescence microscopy on frozen sections of the aortic wall of diverse vertebrates (rat, cow, human and chicken) and by gel electrophoresis of cytoskeletal proteins from whole aortic tissue or from stripped tunica media of cow and man. Most cells of the aortic wall in these species contain vimentin filaments, including smoooth muscle cells of the tunica media. In addition, we have observed aortic cells that are positively stained by antibodies to desmin. The presence of desmin in aortic tissue has also been demonstrated by gel electrophoresis for rat, cow and chicken. In aortic tissue some smooth muscle cells contain both types of intermediate filament proteins, vimentin and desmin. Bovine aorta contains, besides cells in which vimentin and desmin seem to co-exist, distinct bundles of smooth muscle cells, located in outer regions of the tunica media, which contain only desmin. The results suggest that (i) intermediate-sized filaments of both kinds, desmin and vimentin, can occur in vascular smooth muscle in situ and (ii) smooth muscle cells of the vascular system are heterogeneous and can be distinguished by their intermediate filament proteins. The finding of different vascular smooth muscle cells is discussed in relation to development and differentiation of the vascular system.     

In vitro contractility of normal and aneurysmal abdominal aorta muscle coat sections in human and animal material

The objective of the study was to demonstrate spontaneous contractile activity of the smooth muscle coat of the aorta in human and animal material. Spontaneous contractility of smooth muscle tissue, or tonus, is essential for the proper function of many internal organs as observed in the many types of muscle cells which make up the internal structures. The spontaneous contractile activity of the muscle tissue in blood vessels is particularly marked in resistance vessels, regulating circulation within organs or tissues. It can also be observed in large blood vessels such as arteries and veins. The contractile activity of muscular tissue isolated from arteries is the result of a number of factors, including endogenous paracrine substances, neurotransmitters released at postganglionic endings (mostly within the sympathetic system), cells capable of spontaneously generation of functional potentials (pacemaking cells) and the vascular endothelium. Pacemaking cells present in the aortic wall are an important factor in the development of the spontaneous contractility of the muscular coat of the aorta. They are capable of generating functional potentials, resulting in the constant tonus of the smooth muscular coat (comprising the aortic wall) due to tonic contraction. In vitro studies were carried out on abdominal aortic sections collected from 30 New Zealand rabbits with a body mass of 3-4 kilograms each and also on aneurysmal abdominal aortic sections collected during elective aneurysm repair procedures in humans (10 abdominal aortic sections). The 1.5 cm-long sections were mounted in chambers of an automated water bath. The sections were oriented in a transverse and longitudal fashion in order to compare contractility. The incubation medium consisted of Krebs-Henseleit buffer. Spontaneous contractile activity was observed during the study, characterized by rhythmic contractions of the muscular layer of the aorta. The contractile tension within the sections was 0.15 mN in the case of rabbit sections and 0.8 mN in the case of human sections. The average duration of a single contraction was 38.3 +/- 15.05 seconds. The average contraction frequency, i.e. the average number of contractions per minute, was 1.61 +/- 0.54 contractions per minute. The spontaneous contraction is modulated by many factors like endogenous paracrine substances, neurotransmitters or vascular endothelium.     

Smooth muscle cells of blood vessels in response to vibration

In smooth muscle cells of the rat femoral arteries, when the animals have been subjected to a prolonged general vibration (100 Hz, amplitude 0.5 mm), size of their nuclei increases. The nuclei of myocytes in the posterior vena cava in the experimental animals are also larger than in the intact ones. Increase of the volumes of the smooth muscle cells in the posterior vena cava is not connected with the increase of the level of their ploidity. In the control animals accumulations of glycogene are revealed in the smooth muscle cells situating along the course of the internal elastic membrane of the femoral arteries. Single glycogene granules are found in other areas of the t. media. The type of glycogene distribution in the muscle layer of the femoral arteries remains the same in the rats after vibration, however, its general content is essentially decreasing.     

Elastic tissue and smooth muscle volume in elastic and muscular type arteries in the dog

1. Relative elastic tissue and smooth muscle volumes were determined by a stereological point-counting method in arteries with a progressively diminishing diameter, from the aorta towards the periphery. 2. The volume relationship between the smooth muscle cell and its nucleus was determined by the same method. Mean nuclear volume amounted to 6.9% of total smooth muscle cell volume. 3. Relative elastic tissue volume fell from the aorta towards the peripheral arteries, from 22.6% in the ascending aorta to 4--6% in the smallest arteries examined. 4. Relative smooth muscle volume was practically the same and differences between the individual values in the vast majority of arteries examined were non-significant. Total smooth muscle volume, calculated from the volume of the smooth muscle cell nuclei, varied mostly from 45 to 55%. 5. It can be concluded from these results that the ability of small and medium muscular type arteries to change their diameter actively by muscular contraction (as against elastic type arteries, in which this ability is less expressed) is facilitated not only by the organization of the structural components of the arterial wall, but also by the lower elastic tissue volume, which is compensated by the volume of the other passive components of the vascular wall, while relative smooth muscle volume remains the same.     

Arterial intima-media thickness: site-specific associations with HRT and habitual exercise

We determined the site-specific relations of hormone replacement therapy (HRT) and habitual exercise status with intima-media thickness (IMT) in both elastic (carotid) and muscular (femoral) arteries in 77 healthy postmenopausal women: 43 women were sedentary (20 no-HRT and 23 HRT users) and 34 women were endurance trained (14 no-HRT and 20 HRT users). Femoral IMT was not different among the sedentary HRT and endurance-trained no-HRT and HRT groups, but was lower (P < 0.005) in these three groups than in the sedentary no-HRT women. There were no significant group differences in carotid IMT. However, in older women (> or =65 yrs) carotid IMT was smaller (P < 0.05) in HRT compared with no-HRT women. We conclude that both endurance training and HRT status are independently associated with a smaller IMT and these effects are evident primarily in muscular arteries. These results suggest that HRT and habitual exercise may protect postmenopausal women against cardiovascular disease through influences on IMT. The site-specific relations may be due to a greater number of smooth muscle cells and plasticity of muscular arteries compared with elastic arteries and/or differences in heterogeneous influences such as metabolic requirements and hydrostatic pressures.     

Barium responsiveness of the rat aorta and femoral artery during pregnancy

The barium responses of isolated aortic strips and femoral arteries from non-pregnant and pregnant rats were investigated. Barium caused concentration-related increases in tension of vessels from both pregnant and non-pregnant rats. The concentration-response curves of femoral arteries from non-pregnant and 3 week pregnant rats were not different; however contractility and slopes of concentration-response lines for thracic aortas from 1, 2 and 3 week pregnant rats were significantly less than those of aortas from non-pregnant rats. In addition, barium caused rhythmic contractions to develop in both femoral arteries and aortas of 3 week pregnant rats more frequently than vessels from non-pregnant rats. Rhythmic contractions did not develop in aortas from 3 week pregnant rats rats in calcium-free Krebs. Since the effects of barium on the electrical and mechanical activity of various muscles have been postulated to be similar to and/or dependent on calcium, these results may indicate that changes in calcium sensitivity of vascular smooth muscle occur during pregnancy. Such changes may contribute to the blood flow redistribution and other cardiovascular adaptations of pregnancy.     

A comparison of the size of fenestrations in the internal elastic lamina of young and old porcine aortas as seen with the scanning electron microscope.

The size of the fenestrations (windows) in the internal elastic lamina (IEL) of arteries may be important in the functioning of the blood vessel wall. The fenestrations are filled with collagen, muscle, and (or) ground substance, which must be removed to make the fenestration visible with the scanning electron microscope. All of the nonelastic components are removed with a hot alkali solution. Our experiments were designed to compare the fenestration size in the IEL of the thoracic aorta of young (6-8 weeks) and old (6-9 months) pigs. A protocol for digestion of young pig tissue was developed and showed that fresh young aortas should be digested in 0.1 M NaOH at 75 degrees C for 2 h and fixed tissue should be digested for 5 h. The average area of the fenestrations for young pig thoracic aortas digested for 2 h was 1.8 +/- 0.29 (SE) microns 2 and for the old pig aortas digested for 2 h was 1.7 +/- 0.11 (SE) microns 2. These values were not significantly different (p greater than 0.05), but the IEL from young pigs appeared rougher than the previously reported smooth IEL of the adult pigs.     

Inverse relationship between connexin43 and desmin expression in cultured porcine aortic smooth muscle cells.

Our previous work has shown that in vascular tissues the elastic medial regions express high levels of the gap junctional protein, connexin43, but low levels of desmin, while the muscular medial regions express low levels of connexin43 but high levels of desmin. It is uncertain, however, whether this regional difference at the tissue level extends down to the level of the individual cell, or reflects an averaged relationship of groups of cells of different connexin43 and desmin expression. The present study has addressed this question using cultured porcine aortic smooth muscle cells. Immunoconfocal microscopic analysis of single-labeled cells showed that while smooth muscle alpha-actin, calponin and vimentin were positively labeled in the majority of medial smooth muscle cells both in intact porcine aorta and corresponding cultured cells, desmin and connexin43 labeling was highly heterogeneous. In the cultured cells, 0.3-0.5% of cells were found to be desmin-positive, and quantitative analysis after double labeling for desmin and connexin43 revealed that the desmin-positive cells were smaller, and contained significantly lower numbers and smaller sizes of connexin43 gap-junctional spots than did desmin-negative cells. Our findings demonstrate that an inverse expression pattern of connexin43 and desmin holds true at the level of the individual cell. This suggests a close relationship between intrinsic phenotypic control and the regulation of connexin43 expression in the arterial smooth muscle cell.     

Calponin and SM22 as differentiation markers of smooth muscle: spatiotemporal distribution during avian embryonic development

Abstract. Calponin and SM 22 are two proteins related in sequence that are particularly abundant in smooth muscle cells. Here, the distribution patterns of calponin and SM 22 were compared with that of other smooth muscle contractile and cytoskeletal components in the avian embryo using immunofluorescence microscopy and immunoblotting. Like myosin-light-chain kinase and heavy caldesmon, both calponin and SM 22 were more or less exclusively found in smooth muscle cells, during embryonic development and in the adult. Labelling of other cell types including striated muscle was not observed. In contrast, tropomyosin, smooth muscle α-actin, filamin and desmin could also be detected in many other cell types in addition to smooth muscles, at least during part of embryonic life. Calponin and SM 22 appeared almost synchronously during the differentiation of all smooth muscle cell populations, though with a slight time difference in the case of the aorta. The appearance of calponin, SM22 and heavy caldesmon was generally delayed in relation to desmin, tropomyosin, smooth muscle α-actin, myosin-light-chain kinase and filamin and a marked increase in abundance of these proteins was observed in the late embryo and in the adult. From these observations we can conclude that both calponin and SM 22 belong to a group of late differentiation determinants in smooth muscle and may constitute convenient and reliable markers to follow the differentiation of most, if not all, smooth muscle cell populations.