An ultrastructural study of mitotic division in differentiated gastric smooth muscle cells

Summary An ultrastructural examination of tissue from the gizzards of chicks just before and just after hatching showed numerous mitotic divisions in the well differentiated and functional smooth muscle. The nuclei in the very elongate, dividing cells were located centrally. The cytoplasm immediately adjacent to the nuclei contained the normal fully differentiated complement of myofilaments. During the active stages of division, after the breakdown of the nuclear membrane, myofilaments were shown to lie between the individual chromosomes. The process of division only occupied a small portion of the long muscle cells; the ultrastructural changes seen appeared similar to those described in other cell types.This work was supported by grants from the National Heart Foundation of Australia and the Australian Research Grants Committee. Part of this study was completed while J.L.S.C. was in receipt of a Queen Elizabeth II Research Fellowship. T. B. was supported by a Commonwealth Postgraduate Award.     

Impact of glafenine hydrochloride on human endothelial cells and human vascular smooth muscle cells: a substance reducing proliferation, migration and extracellular matrix synthesis

The aim of this study was to examine the effects of glafenine hydrochloride (a nonsteroidal anti-inflammatory drug) on proliferation, clonogenic activity, cell-cycle, migration, and the extracellular matrix protein tenascin of human aortic smooth muscle cells (haSMCs) and human endothelial cells (ECs) in vitro.HaSMCs and ECs were seeded in tissue culture flasks. The cells were treated for 4 days with glafenine hydrochloride (10 microM, 50 microM, 100 microM). Half of the treated groups were incubated again with glafenine hydrochloride, the other half received medium free of glafenine hydrochloride every 4 days until day 20. The growth kinetics and clonogenic activity were assessed. Cell cycle distribution was investigated by FACS, migratory ability was evaluated, and effects on extracellular matrix synthesis were assessed by immunofluorescence.Glafenine hydrochloride inhibited the proliferation and clonogenic activity of haSMCs and ECs in a dose-dependent manner. A block in the G2/M phase and a reduction in the G1 phase occurred. The migratory ability of haSMCs was impaired in a dose-dependent manner and the extracellular matrix protein tenascin was reduced. As glafenine hydrochloride has the ability to fully inhibit proliferation and to partially inhibit migration in haSMCs, it could be an interesting substance for further research in the field of restenosis therapy.     

Lipopolysaccharide regulates toll-like receptor 4 expression in human aortic smooth muscle cells

Lipopolysaccharide (LPS) is a potent activator of cells of the immune and inflammatory systems, including macrophages, monocytes, and endothelial cells (EC). Toll-like receptor 4 (TLR4) has been identified as the primary receptor for LPS. Vascular smooth muscle cells (VSMCs) likely contribute significantly to the inflammation induced by low-level LPS in patients who are at risk for atherosclerosis. Previous study indicated that functional TLR4 was present in VSMCs. However, it remains unclear whether low levels of commercial LPS preparations can affect TLR4 expression in early stage. Here Real-time quantitative PCR analysis was used to detect TLR4 mRNA expression; Immunofluorescence, Western blot analysis and flow cytometry were used to examine TLR4 protein expression. It was shown that TLR4 was present in Human Aortic Smooth Muscle Cells (HASMCs). LPS can up-regulate TLR4 mRNA and protein expression in HASMCs in dose- and time-dependent manner. These data indicate that LPS regulate TLR4 expression in HASMCs.     

The characterization of human uterine smooth muscle cells in culture

Primary cultures initiated from normal human uterine endometrium after total enzymatic dissociation contained epithelioid cells and smooth muscle cells. The smooth muscle cells were subsequently isolated by differential trypsinization and grown in culture for 36 +/- 4 generations. Ultrastructural examination of log and post-confluent cultures of cells at low and high population doubling levels revealed characteristics similar to those of published reports on other smooth muscle cells studied in vivo and in vitro. Among the common features present were: (a) abundant bundles of 60--70 A myofilaments; (b) branched mitochondria; (c) stacks of cisternae of rough endoplasmic reticulum; (d) caveolae intracellulares; (e) nexuses. Other features included ovoid nuclei, a well developed Golgi apparatus and abundant free ribosomes. The subcultured cells exhibited features of dedifferentiation in the log phase of growth and at post-confluency. However, the post-confluent cells showed characteristics indicating redifferentiation back towards their in vivo morphology. Smooth muscle cells isolated from endometrial curettings may provide a useful model for biochemical and pharmacological studies of a cell type derived from a hormonal target tissue as the cells "age" in culture.     

Morphology of smooth muscle cells in the rat thoracic duct

Summary The three-dimensional cytoarchitecture and ultrastructure of the smooth muscle cells in the wall of the rat thoracic duct were investigated by scanning and transmission electron microscopy. The muscle layer basically consists of a single layer of circularly arranged cells. The smooth muscle cell is fusiform or ribbon-like in shape, as in veins or venules with a similar or smaller diameter. Connections by spinous processes are observed between adjacent muscle cells along their length. Spot-like membrane contacts frequently occur in areas where facing membranes are closely apposed. These are thought to be gap junctions and may be responsible for electrical coupling and mechanical attachment. Large invaginations arranged regularly in rows on the surface of the smooth muscle cells can be observed. These invaginations are closely associated with a flattened sarcoplasmic reticulum, and caveolae tend to open into the invaginations.     

Ex vivo generation of mature and functional human smooth muscle cells differentiated from skeletal myoblasts

We described the ex vivo production of mature and functional human smooth muscle cells (SMCs) derived from skeletal myoblasts. Initially, myoblasts expressed all myogenic cell-related markers such as Myf5, MyoD and Myogenin and differentiate into myotubes. After culture in a medium containing vascular endothelial growth factor (VEGF), these cells were shown to have adopted a differentiated SMC identity as demonstrated by alphaSMA, SM22alpha, calponin and smooth muscle-myosin heavy chain expression. Moreover, the cells cultured in the presence of VEGF did not express MyoD anymore and were unable to fuse in multinucleated myotubes. We demonstrated that myoblasts-derived SMCs (MDSMCs) interacted with endothelial cells to form, in vitro, a capillary-like network in three-dimensional collagen culture and, in vivo, a functional vascular structure in a Matrigel implant in nonobese diabetic-severe combined immunodeficient mice. Based on the easily available tissue source and their differentiation into functional SMCs, these data argue that skeletal myoblasts might represent an important tool for SMCs-based cell therapy.     

Viscoelastic properties of isolated rat colon smooth muscle cells

The measurement of the biomechanical properties of gastrointestinal smooth muscle cells is important for the basic understanding of digestive function and the interaction of muscle cells with the matrix. Externally applied forces will deform the cells depending upon their mechanical properties. Hence, the evoked response mediated through stretch-sensitive ion-channels in the smooth muscle cell membrane will depend upon membrane properties and the magnitude of the external force. The aim of this study was to test the hypothesis that gastrointestinal smooth muscle cells behave in a viscoelastic manner. Smooth muscle cells were dissociated from the muscle layers of the descending colon. The viscoelastic properties of the isolated cells were characterized by measuring the mechanical deflection response of the cell membrane to a negative pressure of 1cm H(2)O applied across the cell through a micropipette and fitting the response to a theoretical viscoelastic solid model. The viscoelastic mechanical constants of the isolated cells (N=9) were found to be as follows: k(1)=19.99+/-2.86 Pa, k(2)=7.19+/-1.21 Pa, mu=25.36+/-6.14 Pas and tau=4.84+/-0.95 s. This study represents, to the best of our knowledge, the first quantitative mechanical properties of isolated living smooth muscle cells from the gastrointestinal tract. The mechanical properties determined in this study will be of use in future analytical and numerical smooth muscle cell models to better predict the mechanism between the magnitude of mechanical stimuli, mechanosensitivity and the evoked afferent responses.     

Microvessel precursor smooth muscle cells express head-inserted smooth muscle myosin heavy chain (SM-B) isoform in hyperoxic

The present study analyzes smooth muscle myosin heavy chain (SMMHC) expression as lung microvascular precursor smooth muscle cells (PSMCs), cells derived from fibroblasts and intermediate cells (immature SMCs), acquire a smooth muscle phenotype in anin vivo model of pulmonary hypertension (PH). Because of the unique contractile properties of the SMMHC isoform SM-B, we analyzed its expression in the microvessels (<100 μm diameter) and in larger vessels (100–700 μm) quantitatualy by the labeled [strept]avidin-biotin technique (day 1–28), and related this to cell phenotype by transmission microscopy and protein A-gold labeling (at day 28). Airway SMCs of the normal and hypertensive lung uniformly expressed SM-B whereas vascular SMC expression was heterogeneous. Thus, in some large arteries (and veins) SMCs contained cells expressing SM-B while in others all the cells were immunonegative. Microvascular cells expressing SM-B (arteries and veins) were rare in normal lung and numerous in PH, increasing as wall muscle developed in smaller segments with time. As in large vessels, some microvessels had immunopositive cells and others only negative ones. Ultrastructural analysis confirmed that the SMCs of bronchial vessels, and the septal SMCs adjoining alveolar ducts, contained dense filament arrays decorated with SM-B. While the PSMC processes of the normal lung contained sparse filaments decorated with SM-B, these cells expressed dense filament arrays in PH. Fibroblasts migrating to align around the microvessels also expressed SM-B but in the absence of a filament network. For the first time,we demonstrate in vivo that newly developed microvascular PSMCs express the SMMHC SM-B isoform in PH. Received: 9 April 1998 / Accepted: 9 September 1998     

Fructose-induced peroxynitrite production is mediated by methylglyoxal in vascular smooth muscle cells

Methylglyoxal (MG), a highly reactive molecule, has been implicated in the development of insulin resistance. We investigated whether fructose, a precursor of MG, induced ONOO(-) generation and whether this process was mediated via endogenously increased MG formation. Fructose significantly increased MG generation in vascular smooth muscle cells (VSMCs) in a concentration and time dependent manner. The intracellular production of MG was increased by 153+/-23% or 259+/-28% after cells were treated 6 h with fructose (15 mM or 30 mM), compared with production from untreated cells (p<0.01, n=4 for each group). A significant increase in the production of ONOO(-), NO, and O(2)(*-), was found in the cells treated with fructose (15 mM) or MG (10 microM). Fructose- or MG-induced ONOO(-) generation was significantly inhibited by MG scavengers, including reduced glutathione or N-acetyl-l-cysteine, and by O(2)(*-) or NO inhibitors, such as diphenylene iodonium, superoxide dismutase or N-nitro-l-arginine methyl ester. Moreover, an enhanced iNOS expression was also observed in the cells treated directly with MG which was significantly inhibited when co-application with N-acetyl-l-cysteine. Our results demonstrated that fructose is capable of inducing a significant increase in ONOO(-) production, which is mediated by an enhanced formation of endogenous MG in VSMCs.     

Distinction between smooth muscle,fibroblasts and endothelial cells in culture by the use of fluoresceinated antibodies against smooth muscle actin

Summary FITC-labelled antibodies against native actin from chicken gizzard smooth muscle (Gröschel-Stewart et al., 1976) have been used to stain cultures of guinea-pig vas deferens and taenia coli, rabbit thoracic aorta, rat ventricle and chick skeletal muscle. The I-band of myofibrils of cardiac muscle cells and skeletal muscle myotubes stains intensely. In isolated smooth muscle cells, the staining is located exclusively on long, straight, non-interrupted fibrils which almost fill the cell. Smooth muscle cells which have undergone morphological dedifferentiation to resemble fibroblasts with both phase-contrast microscopy and electronmicroscopy still stain intensely with the actin antibody. In those muscle cultures which contain some fibroblasts or endothelial cells, the non-muscle cells are not stained with the actin antibody even when the reactions are carried out at 37° C for 1 h or after glycerination. Prefusion skeletal muscle myoblasts also do not stain with this antibody.It is concluded that the actin antibody described in this report is directed against a particular sequence of amino acids in muscle actin which is not homologous with non-muscle actin. The usefulness of this antibody in determining the origin of cells in certain pathological conditions such as atherosclerosis is discussed.This work was supported by the Life Insurance Medical Research Fund of Australia and New Zealand, the National Heart Foundation of Australia, the Deutsche Forschungsgemeinschaft and the Wellcome Trust (London). We thank Janet D. McConnell for excellent technical assistance     

An ultrastructural study of regeneration of minced smooth muscle in the vas deferens of the guinea-pig

Summary Electron microscopic studies were made of the regeneration of minced smooth muscle of the vas deferens of the guinea-pig 3 days to 15 weeks after operation. At 3–5 days the mince contained degenerating smooth muscle cells and dedifferentiating cells showing characteristics of embryonic smooth muscle cells: numerous free ribosomes, well developed rough endoplasmic reticulum and Golgi apparatus with few peripherally placed myofilaments associated with dense bodies. During the first two weeks of regeneration, scattered cells surrounded by debris and collagen were separated by a large extra-cellular space. After three weeks, extracellular space was reduced to near normal values. Regenerating cells had a shorter length than normal cells, but during later stages of regeneration they showed an increase in diameter. Muscle effector bundles began to form after 2 to 3 weeks. Initially there were large gaps between the muscle cells, but at later stages of bundle formation, the extracellular space between the muscle cells was much reduced. From 3 weeks, arterioles appeared between the smooth muscle bundles in the regenerating areas. Regeneration of individual smooth muscle cells was complete by 15 weeks after the operation.This work was supported by grants from the Wellcome Trust and the Medical Research Council     

Improved arterial wall model by coculturing vascular endothelial and smooth muscle cells

We have constructed an in vitro arterial wall model by coculturing bovine arterial endothelial cells (ECs) and smooth muscle cells (SMCs). When ECs were seeded directly over SMCs and cocultured in an ordinary culture medium, ECs grew sparsely and did not form a confluent monolayer. Addition of ascorbic acid to the culture medium at concentrations greater than 50 μg/ml increased the production of type IV collagen by the SMCs, and ECs formed a confluent monolayer covering the entire surface of SMCs. Histological studies showed that the thickness of the cell layer composed of ECs and SMCs increased with increasing duration of coculture. This arterial wall model, prepared by our method, may serve as a simple and good in vitro model to study the effects of factors such as biological chemicals and shear stress on cell proliferation and other physiological functions of arterial walls.     

心肌细胞和血管平滑肌细胞收缩调控机制的研究进展

心脏和血管构成体内的心血管系统,两者都具有收缩性。心脏收缩要求在很短的时间内升高室内压,因此要求细胞收缩快速和有力,这就需要细胞的收缩结构和钙调控过程能满足其要求。血管收缩缓慢而持久,其收缩结构及机制也正好与之功能相适应。本文从细胞水平讨论了心脏和血管的收缩结构和收缩机制,以及钙调控机制,并分别对两者之间的异同点作了介绍。     

Altered response of vascular smooth muscle cells to exogenous biochemical stimulation in two- and three-dimensional culture

Removal of vascular smooth muscle cells (SMC) from their native environment alters the biochemical and mechanical signals responsible for maintaining normal cell function, causing a shift from a quiescent, contractile phenotype to a more proliferative, synthetic state. We examined the effect on SMC function of culture on two-dimensional (2D) substrates and in three-dimensional (3D) collagen Type I gels, including the effect of exogenous biochemical stimulation on gel compaction, cell proliferation, and expression of the contractile protein smooth muscle alpha-actin (SMA) in these systems. Embedding of SMC in 3D collagen matrices caused a marked decrease in both cell proliferation and expression of SMA. The presence of the extracellular matrix modulated cellular responses to platelet-derived growth factor BB, heparin, transforming growth factor-beta1, and endothelial cell-conditioned medium. Cell proliferation and SMA expression were shown to be inversely related, while gel compaction and SMA expression were not correlated. Taken together, these results show that SMC phenotype and function can be modulated using biochemical stimulation in vitro, but that the effects produced are dependent on the nature of the extracellular matrix. These findings have implications for the study of vascular biology in vitro, as well as for the development of engineered vascular tissues.     

Human smooth muscle cell cultures of the stomach morphologic and biochemical studies

Summary Smooth muscle cell cultures were prepared from stomach explants obtained surgically from 10 patients with duodenal ulcer. The cultured cells grew in either overlapping layers in “hills and valleys” or in parallel arrays. The ultrastructure studies showed plasmalemmal vesicles, bundles of myofilaments associated with dense bodies, and gap junctions. The synthesis of contractile proteins illustrated the preponderance of actin on myosin and tropomyosin. The synthesis of contractile proteins in stomach smooth muscle cell cultures is significantly higher than in skin fibroblast cultures, i.e. 20 x higher for myosin, 10 x higher for actin, and 30 x higher for tropomyosin.     

Comparison of vascular smooth muscle cells from adult human,monkey and rabbit in primary culture and in subculture

Summary A method is presented for growing large numbers of pure isolated smooth muscle cells from adult human, monkey, and rabbit blood vessels in primary culture.In the first few days in culture these cells closely resembled those in vivo and could be induced to contract with angiotensin II, noradrenaline and mechanical stimulation. They stained intensely with antibodies against smooth muscle actin and myosin. Fibroblasts and endothelial cells did not stain with these antibodies thereby allowing the purity of each batch of cultures to be monitored. This was consistently found to be better than 99%. The smooth muscle cells modified or dedifferentiated after about 9 days in culture to morphologically resemble fibroblasts. At this stage cells could no longer be induced to contract and did not stain with the myosin antibodies. Intense proliferation of these cells soon resulted in a confluent monolayer being formed at which stage some differentiated characteristics returned. The modification or dedifferentiation process could be inhibited by the presence of a feeder layer of fibroblasts or endothelial cells, or the addition of cAMP to the culture medium.Smooth muscle cells which had migrated from explants in primary culture, and cells in subculture, had morphological and functional properties of dedifferentiated cells at all times.The advantages of differentiated rather than dedifferentiated smooth muscle cells in culture for the study of mitogenic agents in atherosclerosis is discussed.The authors wish to thank Professor H.H. Bentall of the Royal Postgraduate Medical School, Hammersmith Hospital, London, for making available human material, and Dr. S. Zeki of Department of Anatomy, University College London for material from monkeys. We are also extremely grateful to Professor G. Burnstock for the use of his laboratory facilitiesHolder of a John Halliday Travelling Fellowship from the Life Insurance Medical Research Fund of Australia and New ZealandResearch Fellow with the National Heart Foundation of AustraliaSupported by the Deutsche Forschungsgemeinschaft     

PTEN in smooth muscle cells is essential for colonic immune homeostasis

Colonic immune homeostasis is essential for normal gastrointestinal tract functioning. In this study, we report that specific gene targeting of phosphatase and tensin homolog (PTEN) in smooth muscle cells caused age-related colonic lymphoid hyperplasia followed by global immune activation in mice. Beginning at 5 weeks of age, these mutant mice displayed massive neutrophil infiltration in the colonic lamina propria. The gene expression levels of pro-inflammatory cytokines and chemokines, including those code for monocyte chemotactic protein-1 (Mcp-1), stromal cell-derived factor 1α (Sdf-1α), and chemokine (C-C motif) ligand 28 (Ccl-28), were upregulated in the colon. Accordingly, permeability and proliferation of the colonic epithelium was compromised. These abnormalities were alleviated to a great extent when the mutants were crossed with Akt1-null mice, indicating that the pathogenesis was mediated by Akt1 signaling. Our results suggest that in smooth muscle cells, PTEN is crucial for maintaining colonic immune homeostasis.