Behavior Of Smooth Muscle Cells Research Articles

Airways and vascular remodelling in asthma and cardiovascular disease: implications for therapeutic intervention. Held in Napa, California, USA, May 1993

P.K. Jefferey, Structural Changes in Asthma. T. Cocks, Changes in Vascular Reactivity in Artherogenesis: Markers of Endothial Cell Dysfunction Associated with Initiation of the Disease. J.H. Campbell and G.R. Campbell, Vascular Smooth Muscle in Culture and its Relevance to the Study of Arthrogenesis. V. Lindner, In Vivo Investigations of Vascular Smooth Muscle Remodelling. M.J. Karnovsky and E.R. Edelman, Heparin/Heparan Sulphate Regulation of Vascular Smooth Muscle Cell Behaviour. M.I. Kotlikoff, Regulation of Cytoplasmic Calcium in Airway Smooth Muscle Cells: Implications for Growth and Contractility. J.G. Martin, Animal Studies with Drugs Affecting Airway Smooth Muscle Thickening. M. Okazawa, P.D. Pare, J.C. Hogg, and R.K. Lambert, Mechanical Consequences of Remodelling of the Airway Wall. A.J. Woolcock and J.K. Peat, Is Asthma a Reversible Disease? L.A. Laitinen and A. Laitinen, Anti-Inflammatory Therapy of Asthma: Corticosteroids. P. Konig, Anti-Inflammatory Therapy of Asthma: Non-Steroid Disease Modifying Agents. P. Gresele, Design of Large Multicentre Therapeutic Trials. The Relevance of the Cardiovascular Experience to the Design of Long Term Anti-Asthma Trials. Discussion Section. Subject Index.

Simultaneous oscillations in the membrane potential of pig coronary artery endothelial and smooth muscle cells.

1. The effects of tetrabutylammonium (TBA) on the mechanical tension and on the electrical behaviour of endothelial and smooth muscle cells were studied in intact porcine coronary artery strips. 2. Superfusion of strips with TBA (2-20 mM) induced mechanical oscillations, leading to an increase in tonic isometric tension. 3. TBA-induced mechanical oscillations were correlated with fluctuations of the membrane potential of endothelial cells, which were identified by iontophoretic injection of Lucifer Yellow. 4. The endothelial cell membrane potential fluctuations appeared as action potentials or smaller amplitude slow waves, and were synchronized with electrical membrane potential fluctuations of the underlying coronary smooth muscle cells. 5. Oscillations induced by TBA in smooth muscle cells were not affected by removal of the endothelium, and depended on the presence of calcium in the external medium. 6. To our knowledge, this is the first description of action potential-like fluctuations in the endothelium. It is concluded that the oscillations were generated in the smooth muscle and that they propagate to the endothelium. The question of the mode of propagation of the signal is discussed.

In vitro model of the inner parts of a vessel wall with cultured human vascular cells

Transfilter culture systems with enzymatically isolated human vascular cells were established to imitate the morphologic situation of the inner parts of a vessel wall. In transfilter cultures, only smooth muscle cells were seeded on one side of the filter, whereas in transfilter cocultures, smooth muscle cells were cultivated in the presence of confluent or nonconfluent (injured) endothelial cells on the other side of the filter. The filter mimics the porous internal elastic lamina. Fourteen days after seeding, cultures were fixed, embedded in Araldite (Serva, Heidelberg, Germany), and prepared for histologic examination (light microscopy, indirect immunofluorescence staining of von Willebrand factor- and alpha-smooth muscle actin-antigen). In transfilter cultures, smooth muscle cells migrated through the pores to the opposite side of the filter, replicated there, and formed fibromuscular proliferates (two to four layers of smooth muscle cells). The proliferation rates of the smooth muscle cells were similar on both sides of the filter and showed an optimum rate on day 4 in culture as determined by 5-bromo-2'-deoxyuridine labeling. By cocultivating a confluent endothelial cell layer on one side of the filter, migratory activity of smooth muscle cells was inhibited. However, when smooth muscle cells were cultivated together with proliferating endothelial cells (injured state), proliferation of smooth muscle cells was massively stimulated (up to 12 layers of smooth muscle cells). The results indicate that the confluency of the endothelial cells and their proliferation rate influence the migratory and proliferative behavior of smooth muscle cells. The transfilter system may be a suitable model for prescreening of potential antiproliferative and antiarteriosclerotic drugs.

The renin-angiotensin system and extracellular matrix.

A hallmark of vascular disease is the inappropriate proliferative and synthetic behaviour of vascular smooth muscle cells. This phenotypically immature behaviour arises as a consequence of the myocytes undergoing phenotypic conversion and/or clonal proliferation of a "fetal" type of smooth muscle cell preexisting in the vessel wall. De-differentiation and initiation of proliferation is not only induced by endothelial desquamation and acute exposure of smooth muscle cells to platelet-derived mitogens, but also occurs in the uninjured blood vessel. Therefore normal components of the blood vessel are implicit in the pathological process. These include vasoconstrictor peptides, growth factor peptides and extracellular matrix molecules. In vitro and in vivo experimentation has indicated that while some of these compounds individually are only mild stimulators of smooth muscle proliferative metabolism, they may act synergistically to induce robust responses. Here we discuss the effects of the vasoconstrictor peptide angiotensin II, which can be locally generated within the vessel wall itself, on the expression of extracellular matrix molecules in vitro and in vivo. We focus on the angiotensin II-modulated expression of extracellular matrix glycoproteins, e.g. thrombospondin, tenascin, fibronectin and laminin.

Transforming growth factor-beta: Signal transduction via protein kinase C in cultured embryonic vascular smooth muscle cells

Transforming growth factor-beta (TGF-beta), an ubiquitous regulatory peptide, has diverse effects on the differentiation and behavior of vascular smooth muscle cells (VSMC). However, the molecular mechanism through which TGF-alpha exerts its effects remains obscure. We investigated the phosphoinositide/protein kinase C [PKC] signaling pathway in the action of TGF-beta on cultured embryonic avian VSMC of differing lineage: a) thoracic aorta, derived from the neural crest; and b) abdominal aorta, derived from mesenchyme. The second messenger responsible for activation of PKC is sn-1,2-diacylglycerol [DAG]; TGF-beta increased the mass amounts of DAG in the membranes of neural crest-derived VSMC concurrent with translocation of PKC from the soluble to the membrane fraction, but TGF-beta had no effect on the DAG or PKC of mesenchyme-derived VSMC. TGF-beta potentiated the growth of platelet-derived growth factor (PDGF)-treated, neural crest-derived VSMC; but abolished PDGF-induced growth of mesenchymal cells. It is concluded that molecular and functional responses of VSMC to TGF-beta are heterogeneous and are functions of the embryonic lineage of the VSMC.

Inhibition of leukocyte extravasation with a monoclonal antibody to CD18 during formation of experimental intimal thickening in rabbit carotid arteries.

In the rabbit model of electrically induced intimal thickening, the adherence processes of different leukocyte subsets as well as the functional significance of leukocyte invasion in the initial migration of smooth muscle cells (SMCs) into the intima were studied by using monoclonal antibody (MAb) 60.3 (directed to the leukocyte adherence glycoprotein CD18), a known potent inhibitor of leukocyte adhesive functions. In control carotid arteries exposed to two periods of electrical stimulation within 36 hours, leukocytes, including all granulocyte subsets, monocytes, and lymphocytes, invaded the cell-free subendothelium. Concomitantly, SMCs were observed to migrate from the media into the intima. In the MAb 60.3-treated rabbits, however, neutrophil emigration into the stimulated arteries was abolished, whereas mononuclear leukocyte accumulation in the intima was only partially inhibited, indicating a complete CD18-dependent mechanism for neutrophil extravasation and additional receptor-ligand systems for the emigration of mononuclear leukocytes. SMCs moved into the intima despite complete blockage of neutrophils and the reduced accumulation of mononuclear cells within the subendothelium after MAb administration. These results preclude neutrophils as initiators of SMC migration into the intima. The influence of mononuclear cells on the migratory behavior of SMCs in intimal thickening formation, however, needs further elucidation.

Expression of Smooth Muscle α-Actin and the Proliferative Activity of Human Smooth Muscle Cells in Culture Is Influenced by Endothelial-Cell and Fibroblast-Conditioned Medium

The relationship between cell density and the expression of smooth-muscle-specific α-actin (SMA) was studied in cultivated human smooth muscle cells (SMC) by indirect immunofluorescence microscopy. Moreover, the influence of an underlying subconfluent and confluent endothelial cell and fibroblast layer on SMA expression and proliferation of SMC was examined. The results demonstrate that the number of SMC expressing SMA correlates with the cell density, e.g. the higher the cell density the higher the number of SMA-expressing cells. This behaviour of SMC was independent of the age of the culture and could be observed over several subsequently established passages in the form of a periodic SMA expression. While underlying layers of subconfluent endothelial cells and fibroblasts neither influenced the growth rate nor SMA expression of SMC, confluent layers of both cell types retarded the expression of SMA and significantly stimulated the proliferative activity of SMC. The results suggest that the interactions between the three cell types present in the arterial wall have to be taken into account when studying complex pathological processes such as atherogenesis in vitro.

Smooth muscle α-actin is a marker for hair follicle dermis in vivo and in vitro

We have examined the expression of smooth muscle alpha-actin in hair follicles in situ, and in hair follicle dermal cells in culture by means of immunohistochemistry. Smooth muscle alpha-actin was present in the dermal sheath component of rat vibrissa, rat pelage and human follicles. Dermal papilla cells within all types of follicles did not express the antigen. However, in culture a large percentage of both hair dermal papilla and dermal sheath cells were stained by this antibody. The same cells were negative when tested with an antibody to desmin. Overall, explant-derived skin fibroblasts had relatively low numbers of positively marked cells, but those from skin regions of high hair-follicle density displayed more smooth muscle alpha-actin expression than fibroblasts from areas with fewer follicles. 2-D SDS-PAGE confirmed that, unlike fibroblasts, cultured papilla cells contained significant quantities of the alpha-actin isoform. The rapid switching on of smooth muscle alpha-actin expression by dermal papilla cells in early culture, contrasts with the behaviour of smooth muscle cells in vitro, and has implications for control of expression of the antigen in normal adult systems. The very high percentage of positively marked cultured papilla and sheath cells also provides a novel marker of cells from follicle dermis, and reinforces the idea that they represent a specialized cell population, contributing to the heterogeneity of fibroblast cell types in the skin dermis, and possibly acting as a source of myofibroblasts during wound healing.

実験的血管傷害

Proliferation of acortic smooth muscle cells (SMC) is one of the key events in restenosis after percutaneous transluminal coronary angioplasty (PTCA). We studied the growth behavior of SMC after rat aortic injury, and the effect of polyamine synthesis inhibitors on the growth of SMC.3H-thymidine incorporation into SMC in aorta denuded with a balloon catheter started at 25 hours after injury, and maximal incorporation occurred 33 to 37 hours after injury. Transient increase in ODC activity was observed within 8 hours after injury. The polyamine level increased and were maximal at 48 hours after injury. Increased levels of spermidine continued until 7 days after injury. Intimal thickening started 7 days after injury and peaked at 21 days.Administration of α-difluoromethylornithine (DFMO), a specific inhibitor of ODC, prevented the enhancement of ODC activity stimulated by aortic injury. Combined administration of DFMO and methyglyoxal bis (guanylhydrazone) (MGBG), a specific inhibitor of S-adenosylmethionine decarboxylase (SAMDC), inhibited the increase in DNA synthesis as well as the polyamine level in the injured aorta, but had no effect on intimal thickening.

Quantitation of collagen fibril cross-section profiles in aging human veins

The cross-section profiles and the diameter distribution of collagen fibrils were examined quantitatively in normal human internal jugular veins at different ages (first, fifth, and eighth decades). All fibrils showed a regular cross-striation pattern of native-type collagen fibrils irrespective of their cross-section profiles. Irregularly outlined (“dysplastic”) fibrillar profiles were observed among the normally occurring circular crosssection profiles. The frequency of such unusual fibrils significantly increased with age. This increase was more pronounced in the tunica media as compared with the tunica adventitia. In the tunica media diameters of collagen fibrils also generally increased with age. In the tunica adventitia, on the other hand, fibrillar diameters were not significantly altered at different ages. The results of this study indicate that the frequency of both the irregularly outlined fibrillar cross-section profiles and increased fibrillar diameters depends on age in normal vascular walls. Therefore, it is concluded that the occurrence of “dysplastic” fibrils is a physiologic age-related phenomenon rather than a morphologic sign of pathologic alteration of collagen. The higher frequency of irregularly outlined collagen fibrils in the tunica media may indicate a higher and/or altered synthetic behavior of smooth muscle cells when compared with fibroblasts of the tunica adventitia.

HISTOGENESIS OF ARTERIAL INTIMAL HYPERPLASIA AND ATHEROSCLEROSIS

This article briefly reviews the histological evidence for the genesis of intimal hyperplasia and atherosclerosis in arteries. It concentrates upon the origin, structure and behaviour of vascular smooth muscle cells in the intimal (subendothelial) layer. The interactions between these intimal muscle cells, the endothelium and the blood are discussed with particular reference to their role in the histogenesis of intimal hyperplasia and atherosclerosis.

Current fluctuations and oscillations in smooth muscle cells from hog carotid artery. Role of the sarcoplasmic reticulum.

Electrical activity of enzymatically isolated, smooth muscle cells from hog carotid arteries was recorded under current clamp and voltage clamp. Under the experimental conditions, membrane potential usually was not stable, and spontaneous hyperpolarizing transients of approximately 100-msec duration were recorded. The amplitude of the transients was markedly voltage dependent and ranged from about 20 mV at a membrane potential of 0 mV to undetectable at membrane potentials negative to -60 mV. Under voltage clamp, transient outward currents displayed a similar voltage dependency. These fluctuations reflect a K+ current; they were abolished by 10 mM tetraethylammonium chloride, a K+ channel blocker, and the current fluctuations reversed direction in high extracellular K+ concentration. Modulators of intracellular Ca2+ concentration also affected electrical activity. Lowering intracellular Ca2+ concentration by addition of 10 mM EGTA to the pipette solution or suppressing sarcoplasmic reticulum function by superfusion with caffeine (10 mM), ryanodine (1 microM), or histamine (3-10 microM) blocked the rapid voltage and current spikes. However, caffeine and histamine induced a much slower hump of outward current before blocking the rapid spikes. This slower transient outward current could be elicited only once after external Ca2+ was removed and is consistent with an activation of K+ channels by Ca2+ released from internal stores. In contrast, removal of external Ca2+ alone failed to abolish the rapid spikes. These results suggest that 1) a Ca2+-dependent K+ conductance can markedly affect the electrical behavior of arterial smooth muscle cells and 2) internal Ca2+ stores, probably the sarcoplasmic reticulum, can support rapid and frequent releases of Ca2+. Exposure to a low concentration of histamine (3 microM) caused synchronization of the irregular, rapid fluctuations giving rise to slow, periodic oscillations of Ca2+-activated K+ conductance with a frequency of 0.1-0.3 Hz. These regular oscillations are reminiscent of periodic Ca2+-induced Ca2+ release, were inhibited by 10 mM caffeine, and point to a modulation of sarcoplasmic reticulum Ca2+ release by histamine.

Culture of human neoplastic gastrointestinal smooth muscle cells.

Due to limited growth potential of primary cultures and the absence of continuous lines of healthy enteric smooth muscle, we have studied the culture behavior of neoplastic gastrointestinal smooth muscle cells. Forty-six human enteric smooth muscle neoplasms (leiomyomas and leiomyosarcomas) were studied while fresh and/or after culture in vitro and growth in vivo in athymic nude mice, with assessments made of morphology, growth characteristics, and biochemical markers of differentiation. The state of differentiation of the tumors varied, with well-differentiated tumors tending to express binding sites for the gastrointestinal hormone cholecystokinin, whereas less well-differentiated tumors did not. Poorly differentiated tumors were the easiest to establish in culture in vitro and to grow in vivo in nude mice. When the cells placed directly into culture proliferated to confluent density, they underwent morphologic differentiation from a spread, fibroblastlike shape to a slender spindle morphology, with these cells possessing fewer biosynthetic organelles and arranging themselves in characteristic "hill and valley" arrays. However, the highly differentiated characteristics of expression of desmin or cholecystokinin-binding sites were not observed in cultured cells. In contrast, cells that had been passaged in nude mice before culture displayed a proliferative phenotype and failed to undergo morphologic differentiation on reaching confluent density. Four human enteric smooth muscle cell lines (documented by chromosomal analysis) originating in stomach, jejunum, ileum, and rectum were established using this strategy.

Culture behavior of healthy bovine gallbladder muscularis smooth muscle cells.

In contrast to the extensive experience culturing vascular smooth muscle cells, little is known about the culture behavior of gastrointestinal smooth muscle cells. In this work, we have studied smooth muscle cells from bovine gallbladder muscularis in culture. Properties reflecting their state of differentiation, including cellular morphology, multicellular arrangements, intermediate filament protein expression, and content of contractile proteins, were studied after dispersed cell preparations were placed into short- and long-term culture on a variety of substrates. Immunocytochemical analysis of intact healthy gallbladder wall demonstrated that the muscularis smooth muscle cells express actin and the intermediate filament protein desmin, while being vimentin-negative. In this tissue they are, however, surrounded by sheets of vimentin-positive fibroblasts. Optimal microdissection of mucosa and serosa from the muscularis therefore still produced a combination of smooth muscle cells and fibroblasts; however, multiple strategies for enriching the yield and purity of muscularis smooth muscle cells for culture were partially successful. Like vascular smooth muscle cells in culture, these visceral smooth muscle cells rapidly underwent morphological dedifferentiation, losing their contactile phenotype. By 5 days in culture, the desmin-positive muscle cells took on a spread, fibroblast-like morphology, likely representing modulation to a proliferative, dedifferentiated state. After long-term culture, the muscle cells were observed to regain some markers of differentiation, but they were never observed to attain complete morphological and functional redifferentiation.

Lack of inhibition of DNA synthesis by prostaglandin I 2 in cultured intimal smooth muscle cells from rabbits

The negative control system for proliferation by prostaglandin I 2 (PGI 2) was studied in the smooth muscle cells (SMC) cultured from the thickened intima (intimal SMC) of rabbit aortas. Indomethacin was found to enhance DNA synthesis of medial SMC but not that of intimal SMC. Exogenously added PGI 2 or its stable analogue, CS-570, was observed to inhibit DNA synthesis of medial SMC enhanced by indomethacin but not that of intimal SMC. These results indicate that medial SMC are negatively controlled by endogenous PGI 2 and that intimal SMC have no such negative control system for cell proliferation. This lack of negative control may be one of the mechanisms underlying the rapid growth behavior of intimal SMC as compared to medial SMC.

Thermodynamic aspects of cell spreading on solid substrata

To verify the validity of thermodynamic approaches to the prediction of cellular behavior, cell spreading of three different cell types on solid substrata was determined in vitro. Solid substrata as well as cell types were selected on the basis of their surface free energies, calculated from contact angle measurements. The surface free energies of the solid substrata ranged from 18-116 erg cm-2. To measure contact angles on cells, a technique was developed in which a multilayer of cells was deposited on a filter and air dried. Cell surface free energies ranged from 60 erg cm-2 for fibroblasts, and 57 for smooth muscle cells, to 91 for HeLa epithelial cells. After adsorption of serum proteins, cell surface free energies of all three cell types converged to approx 74 erg cm-2. The spreading of these cell types from RPMI 1640 medium on the various solid substrata showed that both in the presence and in the absence of serum proteins in the medium, cells spread poorly on low energy substrata (Ys less than 50 erg cm-2), whereas good cell spreading was observed on the higher energy substrata. Calculations of the interfacial free energy of adhesion (delta Fadh) show that delta Fadh decreases with increasing Ys, and equals zero around 45 erg cm-2 for all three cell types in the presence of serum proteins and for HeLa epithelium cells in the absence of serum proteins. This explains the spreading of these cells on the various substrata upon a thermodynamic basis. The results clearly show that substratum surface free energy has a predictive value with respect to cell spreading in vitro, both in the presence and absence of serum proteins. It is noted, however, that interfacial thermodynamics fail to explain the behavior of fibroblasts and smooth muscle cells in the absence of serum proteins, most likely because of the relatively high surface charges of these two cell types.

A review of the histologic changes in vein-to-artery grafts, with particular reference to intimal hyperplasia.

This article reviews the success of vein-to-artery grafts and the published data on patency rates and the major causes for graft failure, ie, intimal hyperplasia and atherosclerosis. It concentrates on the histogenesis of intimal hyperplasia and describes the histologic changes that occur in a vein graft after its insertion. The origin and behavior of intimal smooth-muscle cells are discussed in detail, with particular reference to their role in intimal hyperplasia. A brief experimental section is included to show the specific identification of vein-graft intimal smooth-muscle cells using light-microscopic histochemistry and electron microscopy.

Heparin modulates human intestinal smooth muscle cell proliferation, protein synthesis, and lattice contraction

The effect of heparin on human intestinal smooth muscle cell proliferation, collagen and noncollagen protein synthesis, and collagen lattice contraction was studied in vitro. Proliferation of serumstimulated cells was inhibited in a concentrationrelated fashion by continuous exposure to heparin. The inhibition of proliferation was reversible when heparin was removed from the culture medium. Collagen synthesis was inhibited by 24-h exposure to heparin, but only during that phase of culture (8–12 days) when collagen synthesis was maximal. Noncollagen protein synthesis was down-regulated by 24-h exposure to heparin at all phases of culture tested (5–21 days). Heparin also abolished the contraction of collagen lattices populated by human intestinal smooth muscle cells. These studies demonstrate that heparin plays a significant role in the modulation of human intestinal smooth muscle cell behavior in vitro and suggest that a similar role is played by heparinlike components of the extracellular matrix in vivo.

Analysis of the glycopeptides derived from the extracellular matrix secreted by cultured bovine aortic smooth muscle cells.

Modulation of smooth muscle cell behaviour in culture has been associated with changes in the extracellular matrix. In the present study cultures of bovine aortic smooth muscle cells were compared in the rapidly proliferating and confluent phases of growth. The extracellular matrix was similar in both phases of growth and consisted of glycoproteins ranging from molecular weight 20,000 to over 200,000. The glycopeptides derived from these components displayed several differences. N-linked heteropolysaccharides of the biantennary and complex (more than two branches) types were predominant in the matrix of the confluent phase. Larger amounts of high mannose glycopeptides were present in the preparations from proliferating cells. O-Glycosidic glycopeptides were minor components in both preparations, but a slight increase was noted in the confluent phase of growth. Some of the changes in glycopeptides were interpreted in terms of the levels of the major components of the matrix such as the interstitial procollagens and fibronectin. The results indicate that processing of oligosaccharides associated with secreted glycoproteins of the extracellular matrix correlates with the state of growth of smooth muscle cells in culture.

Phenotype modulation in primary cultures of smooth-muscle cells from rat aorta: Synthesis of collagen and elastin

Early in primary culture, arterial smooth-muscle cells undergo a transition from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and of contractility. At the same time, an extensive rough endoplasmic reticulum and a large Golgi complex are formed, and active synthesis of DNA, RNA and proteins commences. In the present study, chemical and immunocy-tochemical methods were used to investigate the production of extracellular-matrix proteins in relation to this change in phenotypic properties. The results showed that the phase of rapid cellular proliferation that follows the structural modulation of smooth-muscle cells is associated with high rates of collagen and elastin synthesis, as measured by the incorporation of 3H-proline into 3H-hydroxyproline and 3H-valylproline, respectively. SDS-polyacrylamide gel electrophoresis and fluorography indicated that type-I collagen is the main collagen species synthesized by these cells. Smaller amounts of type-V collagen and (although not definitively identified) type-III collagen were also detected. Indirect immunofluorescence and immunoelectron microscopy demonstrated that smooth-muscle cells surround themselves with an incomplete basement membrane, containing laminin and type-IV collagen, and thin fibrils of type-I collagen. Adjacent to these fibrils, aggregates of amorphous, elastin-like material were also found. Our observations confirm and extend earlier notions of a close similarity between the behaviour of arterial smooth-muscle cells during in vitro cultivation and during the early stages of the formation of atherosclerotic lesions.