Article

Analysis of α-smooth muscle actin mRNA expression in rat aortic smooth muscle cells using a specific DNA probe

February 1987
Differentiation 34(3):201-9

February 1987
34(3):201-9

DOI:10.1111/j.1432-0436.1987.tb00067.x

Source
PubMed

Authors:

Giulio Gabbiani

University of Geneva

We constructed two cDNA probes, the first of which hybridizes with all rat actin mRNAs while the second is specific for alpha-smooth muscle (SM) actin mRNA. Northern hybridization using these probes showed that, in normal rat aortic media, the proportion of alpha-SM actin mRNA expression increases during development, reaching about 90% of the total actin mRNA level in adult animals. As compared to the situation in normal aortic media, the proportion of alpha-SM actin mRNA was found to decrease significantly in intimal thickening 15 days after endothelial injury, i.e. when SM cells (SMCs) are actively replicating. At 60 days after injury, the SMCs were observed to have stopped dividing and to have recovered a normal content of alpha-SM actin mRNA. The content of alpha-SM actin mRNA was also selectively decreased (as compared to controls) in the hypotensive abdominal aortic media located below an aortic ligature, while it was not modified in the thoracic hypertensive segment above the same ligature. Primary cultures of rat aortic SMCs synthesize and contain low amounts of alpha-SM actin, but their alpha-SM actin mRNA content is similar to that of SMCs in vivo. As compared to primary cultures, the proportion of alpha-SM actin mRNA was found to be significantly decreased in SMCs at the fifth passage, at which stage it became comparable to the level of synthesized alpha-SM actin. Thus, the synthesis and expression of alpha-SM actin in SMCs appear to be regulated predominantly at the level of gene transcription in certain situations (e.g. aortic ligature in vivo and culture at the fifth passage), and predominantly at a post-transcriptional level in other situations (e.g. primary culture).

Transforming growth factor-β-1 modulates basic fibroblast growth factor induced proteolytic and angiogenic properties of endothelial cells in vitro

Article

Full-text available

Aug 1990

Michael S. Pepper

Tightly controlled proteolytic degradation of the extracellular matrix by invading microvascular en-dothelial cells is believed to be a necessary component of the angiogenic process. We have previously demonstrated the induction of plasminogen activators (PAs) in bovine microvascular endothelial (BME) cells by three agents that induce angiogenesis in vitro: basic FGF (bFGF), PMA, and sodium orthovanadate. Surprisingly , we find that these agents also induce plas-minogen activator inhibitor-1 (PAI-1) activity and mRNA in BME cells. We also find that transforming growth factor-#l (TGF-BI), which in vitro modulates a number of endothelial cell functions relevant to angiogenesis, also increases both PAI-1 and urokinase-type PA (u-PA) mRNA. Thus, production of both proteases and protease inhibitors is increased by an-giogenic agents and TGF-B1. However, the kinetics and amplitude of PAI-1 and u-PA mRNA induction by these agents are strikingly different. We have used the ratio of u-PA:PAI-1 mRNA levels as an indicator of proteolytic balance. This ratio is tilted towards enhanced proteolysis in response to bFGF, towards an-tiproteolysis in response to TGF-B1, and is similar to that in untreated cultures when the two agents are added simultaneously. Using an in vitro angiogenesis assay in three-dimensional fibrin gels, we find that TGF-B1 inhibits the bFGF-induced formation of tube-like structures, resulting in the formation of solid en-dothelial cell cords within the superficial parts of the gel. These results suggest that a net positive proteo-lytic balance is required for capillary lumen formation. A novel perspective is provided on the relationship between extracellular matrix invasion, lumen formation, and net proteolytic balance, thereby reflecting the interplay between angiogenesis-modulating cytokines such as bFGF and TGF-/31.

Transforming growth factor-induced proteolytic and angiogenic properties of endothelial cells in vitro. J. Cell. Biol. 111:743-755

Article

Full-text available

Sep 1990

Tightly controlled proteolytic degradation of the extracellular matrix by invading microvascular endothelial cells is believed to be a necessary component of the angiogenic process. We have previously demonstrated the induction of plasminogen activators (PAs) in bovine microvascular endothelial (BME) cells by three agents that induce angiogenesis in vitro: basic FGF (bFGF), PMA, and sodium orthovanadate. Surprisingly, we find that these agents also induce plasminogen activator inhibitor-1 (PAI-1) activity and mRNA in BME cells. We also find that transforming growth factor-beta 1 (TGF-beta 1), which in vitro modulates a number of endothelial cell functions relevant to angiogenesis, also increases both PAI-1 and urokinase-type PA (u-PA) mRNA. Thus, production of both proteases and protease inhibitors is increased by angiogenic agents and TGF-beta 1. However, the kinetics and amplitude of PAI-1 and u-PA mRNA induction by these agents are strikingly different. We have used the ratio of u-PA:PAI-1 mRNA levels as an indicator of proteolytic balance. This ratio is tilted towards enhanced proteolysis in response to bFGF, towards antiproteolysis in response to TGF-beta 1, and is similar to that in untreated cultures when the two agents are added simultaneously. Using an in vitro angiogenesis assay in three-dimensional fibrin gels, we find that TGF-beta 1 inhibits the bFGF-induced formation of tube-like structures, resulting in the formation of solid endothelial cell cords within the superficial parts of the gel. These results suggest that a net positive proteolytic balance is required for capillary lumen formation. A novel perspective is provided on the relationship between extracellular matrix invasion, lumen formation, and net proteolytic balance, thereby reflecting the interplay between angiogenesis-modulating cytokines such as bFGF and TGF-beta 1.

Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin

Article

Nov 1988

A W Clowes

Quiescent smooth muscle cells (SMC) in normal artery express a pattern of actin isoforms with alpha-smooth muscle (alpha SM) predominance that switches to beta predominance when the cells are proliferating. We have examined the relationship between the change in actin isoforms and entry of SMC into the growth cycle in an in vivo model of SMC proliferation (balloon injured rat carotid artery). alpha SM actin mRNA declined and cytoplasmic (beta + gamma) actin mRNAs increased in early G0/G1 (between 1 and 8 h after injury). In vivo synthesis and in vitro translation experiments demonstrated that functional alpha SM mRNA is decreased 24 h after injury and is proportional to the amount of mRNA present. At 36 h after injury, SMC prepared by enzymatic digestion were sorted into G0/G1 and S/G2 populations; only the SMC committed to proliferate (S/G2 fraction) showed a relative slight decrease in alpha SM actin and, more importantly, a large decrease in alpha SM actin mRNA. A switch from alpha SM predominance to beta predominance was present in the whole SMC population 5 d after injury. To determine if the change in actin isoforms was associated with proliferation, we inhibited SMC proliferation by approximately 80% with heparin, which has previously been shown to block SMC in late G0/G1 and to reduce the growth fraction. The switch in actin mRNAs and synthesis at 24 h was not prevented; however, alpha SM mRNA and protein were reinduced at 5 d in the heparin-treated animals compared to saline-treated controls. These results suggest that in vivo the synthesis of actin isoforms in arterial SMC depends on the mRNA levels and changes after injury in early G0/G1 whether or not the cells subsequently proliferate. The early changes in actin isoforms are not prevented by heparin, but they are eventually reversed if the SMC are kept in the resting state by the heparin treatment.

Expression of smooth muscle alpha‐actin in mesenchymal cells during formation of avian endocardial cushion tissue: A role for transforming growth factor β3

Article

Full-text available

Jul 1997
DEV DYNAM

During early cardiac morphogenesis, outflow tract (OT) and atrio-ventricular (AV) endothelial cells differentiate into mesenchymal cells, which have characteristics of smooth muscle-like myofibroblasts, and which form endocardial cushion tissue, the primordia of valves, and septa in the adult heart. During this embryonic event, transforming growth factor β3 (TGFβ3) is an essential element in the progression of endothelial-transformation into mesenchyme. TGFβs are known to be a potent inducer for mesodermal differentiation and a promoter for differentiation of endothelial cells into smooth muscle-like cells. Using a monoclonal antibody against smooth muscle-specific alpha-actin (SMA), we examined the immunohistochemical staining of this form of actin in avian endocardial cushion tissue formation. To determine whether TGFβ3 initiates the expression of SMA, the pre-migratory AV endothelial monolayer was cultured with or without chicken recombinant TGFβ3 and the expression of SMA was examined immunochemically. Migrating mesenchymal cells expressed SMA beneath the cell surface membrane. These cells showed a reduction of endothelial specific marker antigen, QH1. Stationary endothelial cells did not express SMA. The deposition of SMA in the mesenchymal tissue persisted until the end of the fetal period. Pre-migratory endothelial cells cultured in complete medium (CM199) that contained TGFβ3 expressed SMA, whereas cells cultured in CM199 alone did not. At the onset of the endothelial-mesenchymal transformation, migrating mesenchymal cells express SMA and the expression of this form of actin is upregulated by TGFβ3. The induction of the expression of SMA by TGFβ3 is one of the initial events in the cytoskeletal reorganization in endothelial cells which separate from one another during the initial phenotypic change associated with the endothelial-mesenchymal transformation. Dev. Dyn. 209:296–309, 1997. © 1997 Wiley-Liss, Inc.

Vascular smooth muscle cells exhibit a progressive loss of rigidity with serial culture passaging

Article

Jan 2012
BIORHEOLOGY

One drawback of in vitro cell culturing is the dedifferentiation process that cells experience. Smooth muscle cells (SMC) also change molecularly and morphologically with long term culture. The main objective of this study was to evaluate if culture passages interfere in vascular SMC mechanical behavior. SMC were obtained from five different porcine arterial beds. Optical magnetic twisting cytometry (OMTC) was used to characterize mechanically vascular SMC from different cultures in distinct passages and confocal microscopy/western blotting, to evaluate cytoskeleton and extracellular matrix proteins. We found that vascular SMC rigidity or viscoelastic complex modulus (G) decreases with progression of passages. A statistically significant negative correlation between G and passage was found in four of our five cultures studied. Phalloidin-stained SMC from higher passages exhibited lower mean signal intensity per cell (confocal microscopy) and quantitative western blotting analysis showed a decrease in collagen I content throughout passages. We concluded that vascular SMC progressively lose their stiffness with serial culture passaging. Thus, limiting the number of passages is essential for any experiment measuring viscoelastic properties of SMC in culture.

Spice up the hypertension diet - Curcumin and piperine prevent remodeling of aorta in experimental L-NAME induced hypertension

Article

Full-text available

Oct 2011
NUTR METAB

ABSTRACT: Increase of blood pressure is accompanied by functional and morphological changes in the vascular wall. The presented study explored the effects of curcuma and black pepper compounds on increased blood pressure and remodeling of aorta in the rat model of experimental NO-deficient hypertension. Wistar rats were administered for 6 weeks clear water or L-NAME (40 mg/kg/day) dissolved in water, piperine (20 mg/kg/day), curcumin (100 mg/kg/day) or their combination in corn oil by oral gavage. The systolic blood pressure was measured weekly. Histological slices of thoracic aorta were stained with hematoxylin and eosin, Mallory's phosphotungstic acid hematoxylin (PTAH), orcein, picrosirius red and van Gieson staining and with antibodies against smooth muscle cells actin. Microscopic pictures were digitally processed and morphometrically evaluated. The increase of blood pressure caused by L-NAME was partially prevented by piperine and curcumin, but the effect of their combination was less significant. Animals with hypertension had increased wall thickness and cross-sectional area of the aorta, accompanied by relative increase of PTAH positive myofibrils and decrease of elastin, collagen and actin content. Piperine was able to decrease the content of myofibrils and slightly increase actin, while curcumin also prevented elastin decrease. The combination of spices had similar effects on aortic morphology as curcumin itself. Administration of piperine or curcumin, less their combination, is able to partially prevent the increase of blood pressure caused by chronic L-NAME administration. The spices modify the remodeling of the wall of the aorta induced by hypertension. Our results show that independent administration of curcumin is more effective in preventing negative changes in blood vessel morphology accompanying hypertensive disease.

Expression of Embryonic Fibronectin Isoform EIIIA Parallels -Smooth Muscle Actin in Maturing and Diseased Kidney

Article

Full-text available

Jul 1999

SUMMARY In this study we examined if an association exists between expression of an al- ternatively spliced "embryonic" fibronectin isoform EIIIA (Fn-EIIIA) and a -smooth muscle actin ( a -SMA) in the maturing and adult rat kidney and in two unrelated models of glomer- ular disease, passive accelerated anti-glomerular basement membrane (GBM) nephritis and Habu venom (HV)-induced proliferative glomerulonephritis, using immunohistochemistry and in situ hybridization. Fn-EIIIA and a -SMA proteins were abundantly expressed in mes- angium and in periglomerular and peritubular interstitium of 20-day embryonic and 7-day (D-7) postnatal kidneys in regions of tubule and glomerular development. Staining was markedly reduced in these structures in maturing juvenile (D-14) kidney and was largely lost in adult kidney. Expression of Fn-EIIIA and a -SMA was reinitiated in the mesangium and the periglomerular and peritubular interstitium in both models and was also observed in glomerular crescents in anti-GBM nephritis. Increased expression of Fn-EIIIA mRNA by in situ hybridization corresponded to the localization of protein staining. Dual labeling exper- iments verified co-localization of Fn-EIIIA and a -SMA, showing a strong correlation of stain- ing between location and staining intensity during kidney development, maturation, and disease. Expression of EIIIA mRNA corresponded to protein expression in developing and diseased kidneys and was lost in adult kidney. These studies show a recapitulation of the co-expression of Fn-EIIIA and a -SMA in anti-GBM disease and suggest a functional link for these two proteins. (J Histochem Cytochem 47:787-797, 1999)

Coordinate Expression of -Tropomyosin and Caldesmon Isoforms in Association with Phenotypic Modulation of Smooth Muscle Cells

Article

Full-text available

Jun 1997

Isoform diversity of tropomyosin is generated from the limited genes by a combination of differential transcription and alternative splicing. In the case of the α-tropomyosin (α-TM) gene, exon 2a rather than exon 2b is specifically spliced in α-TM-SM mRNA, which is one of the major tropomyosin isoforms in smooth muscle cells. Here we demonstrate that expressions of α-tropomyosin and caldesmon isoforms are coordinately regulated in association with phenotypic modulation of smooth muscle cells. Molecular cloning and Western and Northern blottings have revealed that in addition to the down-regulation of β-TM-SM, α-TM-SM converted to α-TM-F1 and α-TM-F2 by a selectional change from exon 2a to exon 2b during dedifferentiation of smooth muscle cells in culture. Simultaneously, a change of caldesmon isoforms from high M r type to low M r type was also observed by alternative selection between exons 3b and 4 in the caldesmon gene during this process. In contrast, cultured smooth muscle cells maintaining a differentiated phenotype continued to express α-TM-SM, β-TM-SM, and high M r caldesmon. In situhybridization revealed specific coexpression of α-TM-SM and highM r caldesmon in smooth muscle in developing embryos. These results suggest a common splicing mechanism for phenotype-dependent expression of tropomyosin and caldesmon isoforms in both visceral and vascular smooth muscle cells.

Pericyte growth and contractile phenotype: Modulation by endothelial‐synthesized matrix and comparison with aortic smooth muscle

Article

May 1993
J CELL PHYSIOL

We compared the effects of endothelial-synthesized matrix and purified matrix molecules on pericyte (PC) and aortic smooth muscle cell (SMC) growth, heparin sensitivity, and contractile phenotype in vitro. When PC are plated on endothelial-synthesized (EC) matrix, cell number is, on average, 3.1-fold higher than identical populations grown on plastic. Under the same conditions, SMC proliferation is stimulated 1.6-fold. Purified matrix molecules, such as collagen type IV (COLL) or fibronectin (FN), both major components of the EC matrix, stimulate PC/SMC growth 1.2–1.7-fold. Heparin (100 μg/ml), which inhibits the growth of early passage SMC by 60%, inhibits PC growth ∼50%, when cells were plated on plastic. However, PC plated on EC matrix in the presence of heparin (100 μg/ml) grow as well as parallel cultures grown on plastic (in the absence of heparin). Concomitant with matrix-stimulated proliferation, we observed a marked reduction in PC containing alpha vascular smooth muscle actin (αVSMA), as seen by immunofluorescence using affinity-purified antibodies (173/615 positive pericytes on DOC matrix (28%) vs. 221/285 (77%) positive on glass). SMC respond similarly. Whereas αVSMA protein is markedly altered when PC and SMC are cultured on EC matrix, similar reductions in mRNA are not observed. However, Northern blotting does reveal that PC contain 17–30 times the steady-state levels of αVSMA mRNA compared to SMC. When SMC and PC cultures on plastic are treated with heparin, the steady-state levels of vascular smooth muscle actin mRNA increase 5 and 1.5 fold, respectively. Similarly, heparin treatment of PC grown on plastic induces a 1.8 fold increase in nonmuscle actin mRNA. These heparin-induced alterations in isoactin mRNA levels are not seen when PC are cultured on EC matrix. We also observed reductions in αVSMA and β actin mRNA levels when PC are plated on FN, where they maintain a ratio of 13:1 (α:β). Similar ratios are found in SMC present in rat and bovine aortae in vivo. These steady-state isoactin mRNA ratios are slightly different from those seen in cultured PC (8–10:1; α:β). These results suggest that selective synthesis and remodelling of the endothelial basal lamina may signal alterations in pericyte growth and contractile phenotype during normal vascular morphogenesis, angiogenesis, or during the microvascular remodelling that accompanies hypertensive onset. © 1993 Wiley-Liss, Inc.

Lung myofibroblasts

Article

Jan 1992

Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis: ??-Smooth muscle actin is a marker of mesangial cell proliferation

Article

Full-text available

Apr 1991

Mesangial cell proliferation is common in glomerulonephritis but it is unclear if proliferation is associated with any in vivo alteration in phenotype. We investigated whether mesangial of mesangial proliferative nephritis induced with antibody to the Thy-1 antigen present on mesangial cells. At day 3 glomeruli displayed de novo immunostaining for alpha-smooth muscle actin in a mesangial pattern, correlating with the onset of proliferation, and persisting until day 14. An increase in desmin and vimentin in mesangial regions was also noted. Immunoelectron microscopy confirmed that the actin-positive cells were mesangial cells, and double immunolabeling demonstrated that the smooth muscle actin-positive cells were actively proliferating. Northern analysis of isolated glomerular RNA confirmed an increase in alpha and beta/gamma actin mRNA at days 3 and 5. Complement depletion or platelet depletion prevented or reduced proliferation, respectively; these maneuvers also prevented smooth muscle actin and actin gene expression. Studies of five other experimental models of nephritis confirmed that smooth muscle actin expression is a marker for mesangial cell injury. Thus, mesangial cell proliferation in glomerulonephritis in the rat is associated with a distinct phenotypic change in which mesangial cell assume smooth muscle cell characteristics.

The development expression of the rat alpha-vascular and gamma-enteric smooth muscle isoactins: isolation and characterization of a rat gamma-enteric actin cDNA

Article

Dec 1988

We have isolated and characterized two cDNA clones from whole rat stomach, pRV alpha A-19 and pRE gamma A-11, which are specific for the alpha-vascular and gamma-enteric smooth muscle isoactins, respectively. The rat gamma-enteric smooth muscle actin contains a single amino acid substitution of a proline for a glutamine at position 359 of the mature peptide when compared with the chicken gizzard gamma-actin sequence (J. Vandekerckhove and K. Weber, FEBS Lett. 102:219, 1979). Sequence comparisons of the 5' and 3' untranslated (UT) regions of the two smooth muscle actin cDNAs demonstrate that these regions contain no apparent sequence similarities. Additional comparisons of the 5' UT regions of the two smooth muscle actin cDNAs to all other known actin sequences reveal no apparent sequence similarities for the rat gamma-enteric isoactin within the 15 base pairs of sequence currently available, while the rat alpha-vascular isoactin contains two separate sequences which are similar to sequences within the 5' UT regions of the human and chicken alpha-vascular actin genes. A similar comparison of the 3' UT regions of the two smooth muscle actins demonstrates that the alpha-vascular isoactins do not contain the high degree of cross-species sequence conservation observed for the other isoactins and that the gamma-enteric isoactin contains an inverted sequence of 52 nucleotides which is similar to a sequence found within the 3' UT regions of the human, chicken, and rat beta-cytoplasmic isoactins. These observations complicate the apparent cross-species conservation of isotype specificity of these domains previously observed for the other actin isoforms. Northern blot analysis of day 15 rat embryos and newborn, day 19 postbirth, and adult rats demonstrates that the day 15 rat embryo displays low to undetectable levels of smooth muscle isoactin mRNA expression. By birth, the stomach and small intestine show dramatic increases in alpha-vascular and gamma-enteric actin expression. These initially high levels of expression decrease through day 19 to adulthood. In the adult rat, the uterus and aorta differ in their content of smooth muscle isoactin mRNA. These results demonstrate that the gamma-enteric and alpha-vascular isoactin mRNAs are coexpressed to various degrees in tissues which contain smooth muscle.

Identification and developmental expression of a smooth-muscle gamma-actin in postmeiotic male germ cells of mice

Article

May 1989

Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.

Microscopic and Ultramicroscopic Methods of Identifying and Quantitating Cells in Diffuse Fibrous Arterial Thickenings and Atheromatous Plaque

Chapter

Jan 1991

Identification of the cellular components of atheroma and their distribution within lesions can provide important information concerning pathogenesis of the disease. However, to understand how and why the disease develops, these observations cannot be limited to a single time frame but must be extended over the complete period of development, with particular emphasis on changes in childhood and early adults such as in the PDAY studies described by Professor Robert Wissler (this volume).

Modulation of Fibroblastic Cytoskeletal Features During Wound Healing and Fibrosis

Article

Jan 1995

The mechanisms leading to the retraction of granulation tissue during wound healing have not been presently fully elucidated (for review, see Schürch et al. 1992). Several years ago, our laboratory described the presence within granulation tissue of fibroblasts having several ultrastructural features of smooth muscle (SM) cells, including the presence of microfilament bundles with dense bodies scattered within (Gabbiani and Majno 1972). These cells, called myofibroblasts, have been proposed to play a retractile role in several conditions such as granulation tissue contraction, parenchymal organ retraction, fibromatosis and stromal reaction to epithelial tumors (Sappino et al. 1988, 1990a; Skalli et al. 1989; Darby et al. 1990; Kapanci et al. 1990; Schmitt-Gräff and Gabbiani 1992; for review, see Sappino et al. 1990b). The coincidence of the presence of myofibroblasts with retractile phenomena has supported this hypothesis. However, direct proof of the presence and activity of contractile elements in myofibroblasts has been possible only after suitable techniques have been developed in order to localize and quantify cytoskeletal and contractile proteins within the affected organs. For this purpose, advances in the understanding of cytoskeletal and contractile element morphology and biochemistry in different cells have been of great help (for review, see Skalli and Gabbiani 1988, 1990).

Smooth Muscle Cell and Fibroblast Biological and Functional Features

Chapter

Dec 1995

It is presently more and more accepted that phenotypic features of smooth muscle cells and fibroblasts are not uniform. This plasticity or diversity is clear during development, and, in adults, takes place in normal conditions, resulting in different cytoskeletal features of these cells according to their location; and in pathological situations when smooth muscle cells or fibroblasts react to damaging or inflammatory agents. Generally, the response of each type of cell is stereotyped in that smooth muscle cells assume a synthetic phenotype whereas fibroblasts become more contractile. Both cells tend to proliferate on pathological stimuli. The phenotypic modulation of both cell types corresponds to changes in the expression of cytoskeletal proteins, among which those of intermediate filament proteins as well as those of contractile proteins have been, at least partially, characterized. In smooth muscle cells, the reaction to an endothelial lesion includes the decrease of α-smooth muscle actin and smooth muscle myosin-heavy-chain synthesis (as well as the increase of collagen synthesis), whereas in fibroblasts, tissue injury stimulates α-smooth muscle actin and, albeit exceptionally, smooth muscle myosin-heavy-chain synthesis.

The Role of Arterial Smooth Muscle Cells in the Pathogenesis of Atherosclerosis

Article

Mar 1992

During the development of atherosclerotic lesions in humans or after hypercholesterolemia or deendothelialization in experimental animals, vascular smooth muscle cells (SMCs) proliferate and migrate from the media into the intima. SMCs implicated in this process show a remodeling of morphological and cytoskeletal features, which consists of a loss of microfilament bundles and a switch of actin isoform expression from alpha-smooth muscle to beta-cytoplasmic predominance. Similar features are observed in SMCs of fetal animals, and when SMCs are placed in culture. SMCs show changes of proliferative activity and differentiation upon the stimulation with factors released from platelets, endothelial cells, T lymphocytes and macrophages, such as platelet-derived growth factor, fibroblast growth factor or gamma-interferon. Extracellular matrix components such as heparin can also modulate SMC phenotypic changes. Studies on cell-cell interaction mechanisms mediated by these substances could help in the understanding of atherosclerotic plaque formation and in the planification of strategies for the prevention of this disease.

Expression of phenotype- and proliferation-related genes in rat aortic smooth muscle cells in primary culture

Article

May 1997

Anna Hultgårdh-Nilsson

Objectives: After endothelial injury, smooth muscle cells (SMCs) in the arterial media are modified from a contractile to a synthetic phenotype. This process includes a prominent structural reorganization and makes the cells able to migrate into the intima, divide, and secrete extracellular matrix components. A similar change occurs in culture and the in vitro system has been established as a useful model in which to study the control of SMC differentiation. The purpose of this study was to analyze the expression of a number of phenotype- and proliferation-related genes in vascular SMCs during the first week in primary culture. Methods: SMCs were enzymatically isolated from rat aorta and seeded on substrates of fibronectin (an adhesive plasma protein) and laminin–collagen type IV (two major basement membrane proteins) in a serum-free medium or in uncoated dishes in a serum-containing medium. Total RNA was isolated from the cells after different times of culture and analyzed by Northern blotting for expression of specific gene transcripts. In part, expression of the corresponding proteins was also explored by Western blotting and indirect immunofluorescence microscopy. Results: The results indicate that the proto-oncogenes c-fos, c-jun and c-ets-1 were already activated during the isolation of the cells and then continued to be strongly expressed for a few days. Especially in the serum-free groups, there was also early activation of the genes for the matrix metalloproteinases, stromelysin (MMP-3) and type IV collagenase (MMP-2). In parallel, an increased expression of the genes for two extracellular matrix components was observed, with an early rise in osteopontin mRNA and a later rise in collagen type I mRNA. At the end of the test period, the corresponding proteins were deposited around the cells in a fibrillar pattern. Among the matrix receptors investigated, the β1 integrin subunit showed a high and persistent expression, whereas the α5 and α1 integrin subunits showed lower and more variable mRNA levels. In support of the existence of an autocrine or paracrine platelet-derived growth factor (PDGF) loop, an early rise in expression of the PDGF A-chain gene and a subsequent rise in expression of the PDGF α-receptor gene were noted. Conclusion: It is proposed that the coordinated shift in gene expression here described to take place in connection with the phenotypic modulation of vascular SMCs in primary culture is part of a predetermined genetic program that normally serves the function to engage the cells in a wound healing response.

Effects of transforming growth factor-β1 on human arterial smooth muscle cells in vitro

Article

Jul 1991

S. Bjorkerud

Control of the thickness of the arterial wall is critical, as excessive overgrowth of constituent smooth muscle cells (SMCs) may interfere with blood flow. Effects on SMCs in vitro of several growth factors that are present in blood and/or that are produced endogenously in the arterial wall under certain conditions suggest that influences of endocrine, paracrine, and autocrine nature from stimulating and inhibiting factors may control the smooth muscle tissue mass in the artery. This possibility was explored further by investigating the degree of myodifferentiation in terms of the presence of differentiation-specific filamentous alpha-smooth muscle actin and growth, as measured by the synthesis of DNA and cell number, of SMCs as influenced by their exposure to the mitogens, platelet-derived growth factor and epidermal growth factor, and the bifunctional growth factor, transforming growth factor-beta 1 (TGF-beta 1). Exposure to TGF-beta 1 markedly enhanced differentiation-specific filamentous alpha-smooth muscle actin. This effect did not require arrest of growth, which speaks against a direct causal relation between loss of myodifferentiation (modulation) and multiplication. When quiescent cultures were exposed to TGF-beta 1, alpha-smooth muscle actin was further increased, indicating a more specific differentiation-promoting effect by TGF-beta 1 than mere inhibition of growth. Exposure to TGF-beta 1 also increased spreading, which occurred in parallel with increased filamentous alpha-smooth muscle actin and appearance of stress fibers. Exposure to platelet-derived growth factor under serum-free conditions and to epidermal growth factor in cultures exposed to serum markedly decreased the number of alpha-actin-positive SMCs, indicating a dedifferentiating effect by these mitogens. Exposure of SMCs to TGF-beta 1 under serum-free conditions had pronounced effects on growth, with a concentration-dependent inhibition of platelet-derived growth factor-induced DNA synthesis and cell multiplication. The basal synthesis of DNA in the absence of added growth factors was also greatly inhibited. With serum-free cultures, some loss of cells occurred even with very low concentrations of TGF-beta 1 (5 pg/ml), against which platelet-derived growth factor or a dense cultural state had a protective effect. Enhancement of cell multiplication was not detected for cultivated human SMCs exposed to TGF-beta 1, irrespective of culture density, in contrast to that reported for dense cultures of rat SMCs. TGF-beta 1 is present in and may be released from platelets in situations that promote platelet adherence such as endothelial injury; TGF-beta 1 may also be released from activated macrophages and T lymphocytes either during an immune reaction or inflammation or from the endothelium.(ABSTRACT TRUNCATED AT 400 WORDS)

Chapter 7 Vascular Smooth Muscle Cell Cultures

Article

Dec 1997
METHOD CELL BIOL

Vascular smooth muscle cells (VSMCs) are the predominant cell type in blood vessels of the arterial tree. They are responsible for maintaining vascular tone in response to various hormonal and hemodynamic stimuli and they are the major cell type involved in the process of vascular repair, growth, and response to injury. The development of cell culture systems has greatly aided in this pursuit by providing a controlled environment to study the unique characteristics of these cells in isolation from other influences. When the blood vessel is mechanically injured and its endothelial lining removed, there is significant remodeling of the vessel. Medial VSMCs migrate across the elastic laminae and basement membrane and accumulate in the intima. There they proliferate and secrete increased amounts of interstitial matrix proteins. This new intimal structure is appropriately referred to as the neointima and the cells within it are neointimal VSMCs. This chapter discusses methods for culturing both medial and neointimal VSMCs as well as for culturing VSMCs obtained from atheromatous lesions.

Modulation of actin mRNAs in cultured vascular cells by matrix components and TGF-β 1

Article

Full-text available

May 1989

Summary Alpha-smooth muscle actin is currently considered a marker of smooth muscle cell differentiation. However, during various physiologic and pathologic conditions, it can be expressed, sometimes only transiently, in a variety of other cell types, such as cardiac and skeletal muscle cells, as well as in nonmuscle cells. In this report, the expression of actin mRNAs in cultured rat capillary endothelial cells (RFCs) and aortic smooth muscle cells (SMCs) has been studied by Northern hybridization in two-dimensional cultures seeded on individual extracellular matrix proteins and in three-dimensional type I collagen gels. In two-dimensional cultures, in addition to cytoplasmic actin mRNAs which are normally found in endothelial cell populations, RFCs expressed α-smooth muscle (SM) actin mRNA at low levels. α-SM actin mRNA expression is dramatically enhanced by TGF-β1. In addition, double immunofluorescence staining with anti-vWF and anti-α-SM-1 (a monoclonal antibody to α-SM actin) shows that RFCs co-express the two proteins. In three dimensional cultures, RFCs still expressed vWF, but lost staining for α-SM actin, whereas α-SM actin mRNA became barely detectable. In contrast to two-dimensional cultures, the addition of TGF-β1 to the culture media did not enhance α-SM actin mRNA in three-dimensional cultures, whereas it induced rapid capillary tube formation. Actin mRNA expression was modulated in SMCs by extracellular matrix components and TGF-β1 with a pattern very different from that of RFCs. Namely, the comparison of RFCs with other cell types such as bovine aortic endothelial cells shows that co-expression of endothelial and smooth muscle cell markers is very unique to RFCs and occurs only in particular culture conditions. This could be related to the capacity of these microvascular endothelial cells to modulate their phenotype in physiologic and pathologic conditions, particularly during angiogenesis, and could reflect different embryologic origins for endothelial cell populations.

Smooth muscle actin is expressed in a subpopulation of cultured and cloned fibroblasts and is modulated by ?-interferon

Article

Jul 1992

Clinical and experimental investigations have shown that, during wound healing and fibrocontractive diseases, fibroblasts acquire, more or less permanently according to the situation, morphological and biochemical features of smooth muscle (SM) cells including the expression of α-SM actin. Primary and passaged cultures of rat and human fibroblasts contain a subpopulation of cells expressing α-SM actin. These cells could derive from SM cells and/or pericytes present in the tissue from which cultures have been produced or represent bona fide fibroblasts. We have investigated the presence of α-SM actin in fibroblast cultures, clones, and subclones. In all cases the fibroblastic populations studied showed a proportion of α-SM actin expressing cells. Even after cloning, we never obtained populations negative for α-SM actin. We conclude that α-SM actin expression in fibroblastic cultures is not due to contaminant cells but is a feature of fibroblasts themselves. Our results support the view that fibroblastic cells are a heterogeneous population. It has been previously shown that γ-interferon (γ-IFN) decreases α-SM actin expression in SM cells. In rat and human fibroblasts, γ-IFN decreases α-SM actin protein and mRNA expression as well as proliferation. The properties of this cytokine make it a good candidate for exerting an anti-fibrotic activity in vivo.

Inhibition of rat lipocyte activation in culture by interferon-γ

Article

Sep 1992

Hepatic lipocytes (perisinusoidal, Ito cells) are the primary matrix-producing cells in liver fibrosis. During liver injury they undergo activation, a process characterized by cell proliferation and increased fibrogenesis. We and others have established a culture model in which in vivo features of lipocyte activation can be mimicked by cells grown on plastic. Additionally, we recently showed that activation is associated with new expression of smooth muscle–specific α-actin both in vivo and in culture. Although interferon-γ is known to inhibit collagen production in some systems, its action as a general modulator of lipocyte activation has not been examined; this issue forms the basis for our study.In culture-activated lipocytes, interferon-γ (1,000 U/ml) significantly inhibited lipocyte proliferation as assessed by [3H]thymidine incorporation assay and nuclear autoradiography. In time-course studies of activation, it also markedly reduced expression of smooth muscle–specific α-actin and its messenger RNA. In dose-response experiments, maximal inhibitory effects on smooth muscle–specific α-actin mRNA gene expression were achieved with as little as 10 U interferon-γ/ml. Inhibition of cellular activation was reversible; after interferon-γ withdrawal, messenger RNA levels of smooth muscle–specific α-actin returned to untreated control levels. The effect of interferon-γ extended to extracellular matrix gene expression, with reduction of type I collagen, type IV collagen and total fibronectin messenger RNAs to 3%, 24% and 15% of untreated control levels, respectively. In contrast to the marked effects on smooth muscle-specific α-actin and extracellular matrix gene expression, interferon-γ reduced total protein synthesis by only 17.7%. Moreover, maximal doses of interferon-γ had no effect on cell viability as determined by exclusion of propidium iodide. In summary, interferon-γ is a potent inhibitor of lipocyte activation and may prove useful in the treatment of fibrotic liver injury. (HEPATOLOGY 1992;16:776–784.)

Transcriptional repression of smooth-muscle α-actin gene associated with human papillomavirus type 16 E7 expression

Article

Jul 1995
MOL CARCINOGEN

To explore the role of the E7 viral oncogene from human papillomavirus type 16 (HPV16) in the regulation of cytoskeletal organization, we investigated alterations in particular cytoskeletal components in rat embryonal fibroblasts and three transformants of rat embryonal fibroblast cells produced by transfertions with HPV16 E7 alone (TF1), HPV16 E7 plus adenovirus type 5 E1B (TF3), and HPV16 E7 plus activated Ha-ras (TF4). Marked reductions in smooth-muscle (SM) a-actin content and disrupted organization of stress fibers detected by anti—SM α-actin antibody were evident in all the transformants. These cytoskeletal manifestations were associated with a significant reduction in the mRNAs in these cells. Transcriptional repression by the E7 gene was observed after transient transfection of a chloramphenicol acetyltransferase reporter gene with SM α-actin gene promoter. Nucleotides —123 to —39 of the SM α-actin gene promoter were required for the HPV16 E7 transcriptional repression as shown by the chloramphenicol acetyltransferase assay. The downregulation of this actin isoform mediated by the E7 oncoprotein may play an important role in cell transformation by HPV16. © 1995 Wiley-Liss, Inc.

Interactions of vascular smooth muscle cells with collagen matrices

Article

Dec 1991
Clin Mater

As a first step to producing a viable blood vessel graft, smooth muscle cell (SMC) populated collagen matrices were fabricated with type I collagen extracted from rat tail tendons. The rate at which these matrices were reorganised was determined by the reduction in surface area with time. Porcine and bovine SMC shared an equal ability to contract collagen matrices 1 × 105 cells consistently causing a 99% reduction in upper surface area of a matrix fabricated with 0·5 mg/ml collagen over a period of 26 days. This property did not appear to be affected by the time in culture of the SMC, i.e. passage number. Using a range of collagen concentrations (0·25–1·00 mg/ml), the time to maximum contraction was shown to be proportional to collagen content. Increasing seeding density (5 × 104−5 × 105 per matrix) had the effect of shortening contraction time. The final surface area, however, was not dependent on either of these parameters: all matrices eventually underwent a 98–99% decrease in upper surface area. A histological study highlighted common features indicative of the remodelling process: the formation of cavities, accumulation of cells at the periphery of the matrix, and the formation of collagen fibres.

The response to percutaneous transluminal coronary angioplasty: An ultrastructural study of smooth muscle cells and endothelial cells

Article

Oct 1992
CARDIOVASC PATHOL

We investigated the ultrastructure of the repair tissue formed after percutaneous transluminal coronary angioplasty (PTCA). Four hearts (6 coronary arteries) were investigated; 5 arteries after single PTCA and 1 after repeated PTCA. In the earliest lesion (5 days after PTCA), all smooth muscle cells were of a synthetic phenotype, with abundant rough endoplasmic reticulum and few myofilaments, whereas the oldest lesion (259 days after PTCA) was composed of contractile smooth muscle cells, characterized by abundant myofilaments and small amounts of rough endoplasmic reticulum. In lesions taken at intermediate times (74 and 77 days), the smooth muscle cells were of an intermediate phenotype, with rough endoplasmic reticulum and myofilaments present in approximately equal portions. Regenerating endothelial cells observed at 74 days after PTCA were characterized by a rounded shape and a paucity of intercellular connections in the form of simple junctions, which suggested an immature state. At 259 days after PTCA, the endothelial cells were more mature, as indicated by a flat shape and the presence of many tight junctions and a complete basement membrane. The findings suggest a relation between the stage of endothelial cell regeneration and the phenotype of the smooth muscle cells. Copyright © 1992. Published by Elsevier Inc.

Isolation and Culture of Pulmonary Vascular Smooth Muscle and Endothelial Cells

Chapter

Dec 2011

Experimental quality is directly proportional to cell quality. This might not be a valid mathematical equation, but it very succinctly and elegantly describes the work of biologists from all fields of research: molecular biology or cell biology, proliferation or apoptosis assays, patch clamp electrophysiology or flow cytometry. One element lies at the heart of the success of each of these procedures: viable and healthy cells. Many days and weeks of valuable experimentation time have been spent perfecting cell isolation techniques, simply to guarantee that we can gather reliable data. Although it is possible to purchase cells from various sources, most research groups have developed their own techniques for isolating cells from tissues, techniques which can be adapted relatively easily from one tissue type to another. In addition, although it is always desirable to use freshly isolated cells (less than 8 h after isolation), many investigators have turned to cell culture as a viable alternative to freshly dissociated cells, although this may present some scientific challenges and dilemmas. The current chapter addresses the isolation of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial (PAECs), particularly from humans, rats, and mice. Generally speaking, the methods we outline can also be applied to PASMCs and PAECs from other species, although some modifications may be required. Readers are advised to consult the extensive literature to identify the technique most applicable to their needs.

Transgenic and therapeutic targeting of smooth muscle cells and the challenges presented by phenotypic diversity

Article

Feb 2005

Density‐Dependent modulatioon of vascular smooth muscle α‐actin biosynthetic processing in differntiated BC3H1 myogenic cells

Article

Nov 1992
J CELL BIOCHEM

The expression of vasuclar smooth muscle (VSM) α-actin mRNA during BC3H1 myogenic cell differentiation is specifically stimulated by conditions of high cell density. Non-proteolytic dissociation of cell-cell and cell-matrix contacts in post-confluent cultures of BC3H1 myocytes using EDTA promotes loss of the differentiated morphological phenotype. EDTA-dispersed myocytes exhibit an undifferentiated fibroblastoid appearance and contained reduced levels of both VSM and skeletal α-actin mRNA. Muscle α-actin mRNA levels in EDTA-dispersed myocytes were not restored to that observed in confluent myocyte preparations by experimental manipulation of cell density conditions. Pulse-labeling techniques using L-[35S] cysteine to identify muscle actin biosynthetic intermediates revelated that EDTA-dispersed myocytes expressed nascent forms of both the VSM and skeletal muscle α-actin polypetide chains. However EDTA-dispersed myocytes were less effieicent in the post-translational processing of immautre VSM α-actin compared to non-dispersed myocytes. Simple cell-to-cell contact may mediate VSM α-actin processing efficiency since high-density preparations of EDTA-dispersed myocytes processed more VSM α-actin intermediate than myocytes plated at low density. The actin isoform selectivity of the response to modulation of intercellular contacts suggests that actin biosynthesis in BC3H1 myogenic cells involves mehcanisms capable of discriminating between different isoform classes of nascent actin polypetide chains. © 1992 Wiley-Liss, Inc.

Stromal Reaction to Invasive Cancer: The Cellular Origin of the Myofibroblast and Implications for Tumor Development

Article

Jan 2007
BREAST J

Lone Ronnov-Jessen

Many epithelial malignancies are associated with an extensive conversion of the surrounding normal stroma reminiscent of the transient activation seen during wound healing, albeit in a more chronic form. The most outstanding feature of the altered stroma is the accumulation of peritumoral myofibroblasts. Here, I have focused on the cellular origin of myofibroblasts in human breast cancer, the tumor cell-derived activity responsible for their occurrence, and the possible function of their most pronounced cytoskeletal change. In the field of myofibroblast research, the use of newly developed cell-culture based technologies have been instrumental. Stromal cells in short-term cultures have been thoroughly characterized using markers defined by their staining patterns in the tissue of origin. Efforts to purify non-activated fibroblasts prior to cultivation and the introduction of chemically defined culture conditions have allowed the elements of conversion to be studied at the molecular level. Furthermore, the development of a so called tumor environment assay, in which tumor cell-stromal cell interactions result in functional replicas of typical tumor histology, has proven a powerful tool in the search for the contribution of the individual stromal cell types to the complex scenario of peritumoral myofibroblasts. The cell-cell interactions seen in the tumor environment assay have been validated by in vivo coinoculation of tumor cells and myofibroblasts in nude mice. The most recent data point to the function of fibroblast cytoskeletal conversion in immobilizing the cells in close apposition to tumor cells. The comprehensive participation of normal fibroblasts in malignancies promise for future new therapeutic strategies directed against tumor cell-fibroblast interaction.?

Foam cells from aorta and spleen overexpress apolipoprotein E in the absence of hypercholesterolemia

Article

Mar 1992
BIOCHEM BIOPH RES CO

We have studied Apo E expression in atherosclerotic lesions and spleens in rabbits after a cholesterol-rich diet is discontinued and plasma cholesterol levels return to normal values. After 16 weeks, foam cells are still present in the atherosclerotic lesions and Apo E expression persists restricted solely to the lesion as ascertained by RNA hybridization and northern blot. Apo E expression is induced in spleens of hypercholesterolemic rabbits. However Apo E mRNA levels decrease in this organ parallel to the disappearance of lipid loaded macrophages. These results of in vivo studies indicate that Apo E overexpression in foam cells does not depend on high serum cholesterol levels, but is a characteristic of macrophages that have acquired the foam cell phenotype.

Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features

Article

Jul 2006
DIFFERENTIATION

It is well known that arterial smooth muscle cells (SMC) of adult rats, cultured in a medium containing fetal calf serum (FCS), replicate actively and lose the expression of differentiation markers, such as desmin, smooth muscle (SM) myosin and alpha-SM actin. We report here that compared to freshly isolated cells, primary cultures of SMC from newborn animals show no change in the number of alpha-SM actin containing cells and a less important decrease in the number of desmin and SM myosin containing cells than that seen in primary cultures of SMC from adult animals; moreover, contrary to what is seen in SMC cultured from adult animals, they show an increase of alpha-SM actin mRNA level, alpha-SM actin synthesis and expression per cell. These features are partially maintained at the 5th passage, when the cytoskeletal equipment of adult SMC has further evolved toward dedifferentiation. Cloned newborn rat SMC continue to express alpha-SM actin, desmin and SM myosin at the 5th passage. Thus, newborn SMC maintain, at least in part, the potential to express differentiated features in culture. Heparin has been proposed to control proliferation and differentiation of arterial SMC. When cultured in the presence of heparin, newborn SMC show an increase of alpha-SM actin synthesis and content but no modification of the proportion of alpha-SM actin total (measured by Northern blots) and functional (measured by in vitro translation in a reticulocyte lysate) mRNAs compared to control cells cultured for the same time in FCS containing medium. This suggests that heparin action is exerted at a translational or post-translational level. Cultured newborn rat aortic SMC furnish an in vitro model for the study of several aspects of SMC differentiation and possibly of mechanisms leading to the establishment and prevention of atheromatous plaques.

Colonic pericryptal fibroblasts

Article

Nov 1989

Experimental evidence has shown that fetal gut mesenchymal cells can modulate epithelial cell differentiation. It is postulated that reciprocal stromal-epithelial interactions in the digestive tract are maintained beyond embryonic life. The mature colonic mucosa contains pericryptal fibroblasts (PCF), a stromal cell type exhibiting smooth muscle morphological features, which are thought to regulate the growth and differentiation of adjacent epithelial cells. Using an antibody directed at -smooth muscle actin, which is constantly expressed in smooth muscle cells, we performed an immunohistochemical study on human embryonic tissues to assess PCF differentiation during development. PCF expressing ct-smooth muscle actin were first detected around the 21st week of gestation, at the bases of the crypts; the number of differentiated PCF increased then progressively, in synchrony with epithelial proliferation, to achieve at birth the characteristic distribution found in adults. We analyzed a series of non-malignant and malignant epithelial proliferative lesions of the adult colon by the same technique. Only sparse immunoreactive PCF were observed in 10/10 pure tubular adenomas, whereas in 11/11 villous adenomas immunoreactive PCF were consistently found bordering proliferative epithelia. Interestingly, 3/5 papillary adenomas, associated with areas of moderate to marked dysplasia, demonstrated foci of multi-layered immunoreactive PCF. In 14/14 carcinomas examined, PCF were no longer recognizable; stromal cells expressing variable amounts of -smooth muscle actin, constituting the desmoplastic reaction, were constantly present. These observations establish immunohistochemically a smooth muscle phenotypic feature of PCF, which is acquired at mid-gestation, and the ability of PCF to proliferate in conjunction with some epithelial neoplasias. These findings might help to clarify the histogenesis of PCF and to improve our understanding of the mesenchymal-epithelial interactions suspected to operate during organogenesis as well as benign and malignant neoplastic conditions.

Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

Article

Jun 1989

Human arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed.

Changes in expression and organization of smooth-muscle-specific α-actin during fibronectin-mediated modulation of arterial smooth muscle cell phenotype

Article

Oct 1990

AbstractThe spreading of freshly isolated arterial smooth muscle cells on a substrate of fibronectin is mediated by an integrin receptor on the cell surface. It is associated with organization of actin filaments in stress fibers and marked changes in cell morphology and function, collectively referred to as a transition from a contractile to a synthetic phenotype. To study further how extracellular matrix components affect smooth muscle phenotype, we have analyzed the expression and organization of smooth-muscle-specific α-actin in freshly isolated rat aortic smooth muscle cells cultured on a substrate of fibronectin under serum-free conditions. Northern-blot analysis showed that the expression of mRNA for smooth muscle α-actin, but not for nonmus-cle actin, was strongly repressed during primary culture. On the other hand, the cellular content of α-actin was only moderately changed during the same period. Indirect immunofluorescence staining revealed that non-muscle actin was rapidly organized in stress fibers, which did not stain with a monoclonal antibody against smooth muscle α-actin. Filament bundles containing α-actin were most prominent in the central parts of the cytoplasm and gradually disappeared as the spreading of the cells progressed. In contrast to the situation with nonmuscle actin, there was no apparent overlap in the staining for α-actin and the fibronectin receptor (α5β1), indicating that this receptor interacted with nonmuscle actin during the initial spreading process. Taken together, the results show that the expression and organization of smooth muscle α-actin are changed during interaction of the cells with fibronectin early in primary culture. They support the notion that integrin-mediated interactions between extracellular matrix components and arterial smooth muscle cells take part in the control of smooth muscle phenotype.

Fine ambient air particulate matter exposure induces molecular alterations associated with vascular disease progression within plaques of atherosclerotic susceptible mice

Article

May 2009
INHAL TOXICOL

Epidemiology studies have reported associations between increased mortality and morbidity with exposure to particulate air pollution, particularly within individuals with preexisting cardiovascular disease (CVD). Clinical and toxicological studies have provided evidence that exposure to ambient air particulate matter (PM) impacts CVD by increasing plaque size. It is unclear whether PM-induced increased plaque size is associated with molecular disease progression. This study examines molecular profiles within plaques recovered from ApoE-/- mice exposed to concentrated ambient air particles (CAPs) to determine whether pulmonary deposition of PM contributes to molecular alterations leading to vascular disease progression. Laser capture microdissection was used to recover atherosclerotic plaques from ApoE -/- male mice exposed daily for 5 mo to filtered air or CAPs. Alterations in mRNA expression was assessed in microdissected plaques of CAPs-exposed and air controls using the Affymetrix microarray platform. Bioinformatic analysis indicated alterations in 611 genes: 395 genes downregulated and 216 genes upregulated. Gene ontology revealed CAPs-induced changes to inflammation, proliferation, cell cycle, hematological system, and cardiovascular pathways. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) verified microarray data also revealing gene expression alterations undetected by the microarray analysis, i.e., decreased expression of α-actin for smooth muscle cells, and increased expression of the macrophage marker Cd68 and of β-actin. Comparison of CAPs-induced gene expression profiles demonstrated consistency with previously published gene expression profiles in the ApoE-/- mouse model and humans associated with plaque progression. These results indicate that exposure to fine PM induces molecular alterations associated with vascular disease progression and provides insight into potential biological pathways responsible for this effect.

Development and characterization of a cloned rat pulmonary arterial smooth muscle cell line that maintains differentiated properties through multiple subcultures

Article

Jan 1993

Pulmonary hypertension is associated with abnormal pulmonary arterial contractility and growth. The mechanisms for these abnormalities are largely unknown. To study these processes, we sought to develop an in vitro system. Even though cultured aortic and pulmonary artery smooth muscle cells (SMCs) have been of considerable value in studying smooth muscle biology, one drawback of this system has been that these cells often lose differentiated properties in an unpredictable manner when they are passaged in culture. In addition, there appear to be significant differences in physiological and pathological responses between the systemic and pulmonary circulations, many of which could be directly related to the smooth muscle. We therefore established a cloned population of rat pulmonary arterial SMCs (PASMCs) that maintain differentiated properties through multiple subcultures. PASMCs were obtained initially by enzymatic dissociation from pulmonary arteries of adult Sprague-Dawley rats. From these cells, clones were isolated. The cloned cells retained expression of functional surface receptors for angiotensin II, norepinephrine, and alpha-thrombin and high levels of the smooth muscle isoforms of alpha-actin, myosin heavy chain, myosin regulatory light chain, and alpha-tropomyosin mRNAs even after multiple passages. The cells could also be transfected and processed exogenous transcripts in a smooth muscle-specific fashion. These cloned PASMCs retain many differentiated characteristics and should be valuable for future studies of pulmonary vascular smooth muscle cell biology.

Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features

Article

May 1992

Two cloned cerebral endothelial cell phenotypes: an in vitro model for angiogenesis?

Article

Feb 1992
EXS

Expression of smooth muscle alpha-actin and migratory behaviour of cloned cerebral endothelial cells (cEC) which exhibited two distinct phenotypes (type I, type II) were studied. Removal of mitogenic factors (alpha ECGF, ECGS) and heparin from the culture medium resulted in a smooth muscle-like appearance (type II) of the cells, expression of smooth muscle alpha-actin protein and smooth muscle actin mRNA and in an increased migratory activity. In contrast, addition of growth factors and heparin led to a cobblestone-like phenotype (type I) which lacked the expression of smooth muscle alpha-actin but expressed other proteins as determined by 2-D-gel electrophoresis.

Regulation of Sarco-plasmic reticulum Ca2+-ATPases during platelet-derived growth factor induced smooth muscle cell proliferation

Article

Full-text available

Sep 1992

The role of the sarco-endoplasmic reticulum Ca(2+)-ATPases (SERCA) in the regulation of cell proliferation by Ca2+ was investigated by testing the effect of platelet-derived growth factor (PDGF) on cultured pig aorta smooth muscle cells. For this purpose, the PDGF-mediated rise in the Ca2+ concentration was first examined for its ability to induce the formation of prostaglandins from the specific membrane enzyme, cyclooxygenase. In parallel experiments, similar conditions (10 ng/ml PDGF for 24 h) were used to investigate the smooth muscle cell membrane SERCA2 isoforms. Total SERCA2 activity rose by 472% as reflected by their specific formation of phosphorylated intermediate (E approximately P). This rise correlated with an increase in the amount of SERCA2 proteins (100 kDa) as shown by Western blotting. With isoform-specific anti-SERCA2-a and anti-SERCA2-b antibodies, we demonstrated that the increase in total SERCA2 proteins concerned the minor isoform SERCA2-a, which rose 10-fold, whereas SERCA2-b proteins were not affected. Lastly, Northern blotting using riboprobes showed that PDGF treatment increased the SERCA2-a mRNA species by 82%, and concomitantly decreased the SERCA2-b mRNA by 28%, as a result of isoform switching. We conclude that up-regulation of the SERCA2-type Ca(2+)-ATPases occurs in PDGF-treated smooth muscle cells, which suggests that this enzymatic system plays an essential part in cell proliferation.

O-Acylated heparin derivatives with low anticoagulant activity decrease proliferation and increase α-smooth muscle actin expression in cultured arterial smooth muscle cells

Article

Sep 1992
EUR J PHARMACOL

Selectively O-acylated derivatives of various glycosaminoglycans were prepared and tested in vitro for their anticoagulant activity and their antiproliferative effect on rat and rabbit smooth muscle cells. When O-acylation (butyrylation or hexanoylation) had been performed on periodate-depolymerized heparin fragments having very low anticoagulant activity, the antiproliferative potency was markedly increased (IC50 = 2 and 1 micrograms/ml respectively, versus 31 micrograms/ml for starting compound) without an increase in anticoagulant activity. The antiproliferative activity was related to the degree of acylation. The O-acylated derivatives of heparin fragments were also very active in reversing the de-differentiation of smooth muscle cell in culture, as estimated by the increase in the expression of alpha-smooth muscle actin and alpha-smooth muscle actin mRNA.

Inhibition of rat hepatic lipocyte activation in culture by interferon-??

Article

Oct 1992

Hepatic lipocytes (perisinusoidal, Ito cells) are the primary matrix-producing cells in liver fibrosis. During liver injury they undergo activation, a process characterized by cell proliferation and increased fibrogenesis. We and others have established a culture model in which in vivo features of lipocyte activation can be mimicked by cells grown on plastic. Additionally, we recently showed that activation is associated with new expression of smooth muscle-specific alpha-actin both in vivo and in culture. Although interferon-gamma is known to inhibit collagen production in some systems, its action as a general modulator of lipocyte activation has not been examined; this issue forms the basis for our study. In culture-activated lipocytes, interferon-gamma (1,000 U/ml) significantly inhibited lipocyte proliferation as assessed by [3H]thymidine incorporation assay and nuclear autoradiography. In time-course studies of activation, it also markedly reduced expression of smooth muscle-specific alpha-actin and its messenger RNA. In dose-response experiments, maximal inhibitory effects on smooth muscle-specific alpha-actin mRNA gene expression were achieved with as little as 10 U interferon-gamma/ml. Inhibition of cellular activation was reversible; after interferon-gamma withdrawal, messenger RNA levels of smooth muscle-specific alpha-actin returned to untreated control levels. The effect of interferon-gamma extended to extracellular matrix gene expression, with reduction of type I collagen, type IV collagen and total fibronectin messenger RNAs to 3%, 24% and 15% of untreated control levels, respectively. In contrast to the marked effects on smooth muscle-specific alpha-actin and extracellular matrix gene expression, interferon-gamma reduced total protein synthesis by only 17.7%.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasminogen activator inhibitor-1 is induced in migrating endothelial cells

Article

Oct 1992

It has been proposed that a finely tuned protease-anti-protease equilibrium must be maintained during processes of cell migration in order to limit extracellular proteolysis to the close proximity of the cell surface, and thereby to prevent excessive extracellular matrix degradation. We have previously shown that urokinase-type plasminogen activator (u-PA) activity is induced in microvascular endothelial cells migrating from the edges of a wounded monolayer in vitro (Pepper et al., J. Cell Biol., 105:2535-2541, 1987). By Northern analysis, we now demonstrate that plasminogen activator inhibitor 1 (PAI-1) mRNA is increased in multiple-wounded monolayers of bovine microvascular (BME) or aortic (BAE) endothelial cells, with a maximal 7- and 9-fold increase 4 h after wounding, respectively. By in situ hybridization, we demonstrate that the increase in PAI-1 mRNA is localized to cells at the edge of a wounded BME or BAE cell monolayer. The increase in PAI-1 mRNA observed in BME cells is independent of cell division and is inhibited by antibodies to basic fibroblast growth factor (bFGF), suggesting that PAI-1 induction in migrating cells is mediated by the autocrine activity of bFGF. Taken together with our previous observations on the induction of u-PA, these results support the hypothesis that the proteolytic balance in the pericellular environment of migrating cells is regulated through the concomitant production of proteases and protease inhibitors.

Differential modulation of vascular integrin and extracellular matrix expression in vitro by TGF-??1 correlates with reciprocal effects on cell migration

Article

Oct 1992

In vitro, BAEC and BASMC migratory phenotypes are known to be reciprocally modulated by both soluble factors and extracellular matrix proteins. In addition, integrin matrix receptors mediate endothelial and smooth muscle cell attachment and migration. To further elucidate these phenomena, we studied the effects of TGF-beta 1 on integrin expression by vascular BASMC and BAEC in tissue culture. TGF-beta 1 upregulated mRNA levels and surface pools of BASMC beta 3 integrin classes without modulating beta 1 integrin mRNA levels or expression of beta 1 integrin organization. In contrast to its effects on BASMC, TGF-beta 1 increased BAEC mRNA levels and surface expression of beta 1 and beta 3 integrins without altering their organization. Conversely, extracellular matrix components (fibronectin, laminin, and fibrinogen) organized cell surface integrins in both BASMC and BAEC without affecting the size of their cell surface pools. These data are consistent with the hypothesis that SMC and EC behavior in neointimal lesions may be modulated, in part, through a coordination of soluble factor and extracellular matrix protein regulation of integrin surface expression and organization.

Differential expression of SM1 and SM2 myosin isoforms in cultured vascular smooth muscle

Article

Apr 1992
Am J Physiol

Ribonuclease protection assays were used to measure expression of smooth muscle (SM) specific myosin heavy chain (MHC) isoforms SM1 (204 kDa) and SM2 (200 kDa) and also nonmuscle MHC-A in cultured smooth muscle cells isolated from rat aorta. In cells grown in 10% serum for 3-5 days until subconfluent, SM1 MHC mRNA decreased by 30% and SM2 MHC mRNA decreased by 80%. In cells grown in confluency for 7-11 days, SM1 MHC mRNA decreased by 45% and SM2 MHC mRNA decreased by 80%. Similar reductions were found in passaged cells. Serum withdrawal for 1-2 days from confluent cultures had little or no effect on SM1 or SM2 MHC mRNA levels. In contrast, nonmuscle MHC-A mRNA increased 10-fold in subconfluent cultures but increased only threefold higher than controls in quiescent cells. Myosin protein analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that SM1 MHC protein was detectable at a reduced level in confluent cultured cells, whereas SM2 MHC protein was absent in confluent cells. The decrease in SM2 was much greater than SM1, indicating differential regulation. An apparently new isoform of SM1 MHC migrating with a mobility similar to SM2 type MHC was detected by immunoblot analysis in cultured cells.

Heparan sulfate-degrading enzymes induce modulation of smooth muscle phenotype

Article

Jun 1992

Macrophages cocultured with rabbit aortic smooth muscle cells at a ratio of 1:3 degraded all the 35S-labeled heparan sulfate proteoglycan from the smooth muscle surface into free sulfate (Kav of 0.84 on Sepharose 6B). Concomitantly, the same macrophages induced a decrease in the volume fraction of myofilaments (Vvmyo) of the smooth muscle cells and a decrease in alpha-actin mRNA as a percentage of total actin mRNA. Both macrophage lysosomal lysate at neutral pH and heparinase degraded cell-free 35S-labeled matrix deposited by smooth muscle cells into fragments which eluted at a Kav of 0.63 and which were identified as heparan sulfate chains by their complete degradation in the presence of low pH nitrous acid. At acid pH the macrophage lysosomal lysate completely degraded the heparan sulfate to free sulfate (Kav 0.84). Both macrophage lysosomal lysate and commercial heparinase at neutral pH induced smooth muscle phenotypic change while other enzymes such as trypsin and chondroitin ABC lyase had no effect. It was therefore suggested that the active factor present in the macrophages is a lysosomal heparan sulfate-degrading endoglycosidase (heparinase). Only a small amount of heparan sulfate-degrading activity was released into the incubation medium by living macrophages, and there was no heparinase activity on their isolated plasma membranes, although proteolytic enzymes were evident in both instances. In pulse-chase studies, high Vvmyo smooth muscle cells were seen to constantly internalize and degrade 35S-labeled heparan sulfate proteoglycan from their own pericellular compartment, suggesting that this may be the mechanism by which smooth muscle phenotype is maintained under normal circumstances and that removal of heparan sulfate from the surface of smooth muscle cells and its degradation by macrophages temporarily interrupts this process, inducing smooth muscle phenotypic change.

The Cytoskeleton of Arterial Smooth Muscle Cells during Human and Experimental Atheromatosis

Article

Jul 1992
Kidney Int Suppl

Smooth muscle cells (SMCs) implicated in the human atheromatous process show dedifferentiated features characterized by typical changes of cytoskeletal elements. In the normal media, quiescent SMCs express predominantly the alpha-smooth muscle (SM) actin isoform. In the human atheromatous plaque, and in rat experimental intimal thickening 15 days after balloon-induced endothelial injury, a decrease of the alpha-SM actin isoform and a predominance of the beta-cytoplasmic actin isoform develop. Proliferating SMCs in vivo assume fetal phenotypic features which are also observed in cultured SMCs. Thus, the study of cytoskeletal changes allows a better definition of SMC phenotype. Furthermore, in vitro SMCs may represent a useful experimental model to study phenotypic modifications of SMCs during the pathological process. Cytokines and growth factors, released by cells present in the atheromatous plaque, and extracellular components of the arterial wall, such as heparin, modulate the expression of alpha-SM actin in cultured SMCs and represent good candidates to exert important regulatory actions in vivo. A better understanding of the mechanisms leading to cytoskeletal modifications may help in the clarification of the mechanisms playing a role in the development of arterial pathological events.

Transforming growth factor beta 1 modulates extracellular matrix organization and cell-Cell junctional complex formation during

Article

Jan 1990

Transforming growth factor-beta 1 (TGF-beta 1) is angiogenic in vivo. In two-dimensional (2-D) culture systems microvascular endothelial cell proliferation is inhibited up to 80% by TGF-beta 1; however, in three-dimensional (3-D) collagen gels TGF-beta 1 is found to have no effect on proliferation while eliciting the formation of calcium and magnesium dependent tube-like structures mimicking angiogenesis. DNA analyses performed on 3-D cell cultures reveal no significant difference in the amount of DNA or cell number in control versus TGF-beta 1 treated cultures. In 2-D cultures TGF-beta 1 is known to increase cellular fibronectin accumulation; however, in 3-D cultures no difference is seen between control and TGF-beta 1 treated cells as established by ELISA testing for type IV collagen, fibronectin, and laminin. In 3-D cultures there is increased synthesis and secretion of type V collagen in both control and TGF-beta 1 treated cultures over 2-D cultures. Even though an equal amount of type V collagen is seen in both 3-D conditions, there is a reorganization of the protein with concentration along an organizing basal lamina in TGF-beta 1 treated cultures. EM morphological analyses on 3-D cultures illustrate quiescent, control cells lacking cell contacts. In contrast, TGF-beta 1 treated cells show increased pseudopod formation, cell-cell contact, and organized basal lamina-like material closely apposed to the "abluminal" plasma membranes. TGF-beta 1 treated cells also appear to form junctional complexes between adjoining cells. Immunofluorescence using specific antibodies to the tight junction protein ZO-1 results in staining at apparent cell-cell junctions in the 3-D cultures. Northern blots of freshly isolated microvascular endothelium, 2-D and 3-D cultures, using cDNA and cRNA probes specific for the ZO-1 tight junction protein, reveal the presence of the 7.8 kb mRNA. Western blots of rat epididymal fat pad endothelial cells (RFC) monolayer lysates probed with anti-ZO-1 label a 220 kd band which co-migrates with the bonafide ZO-1 protein. These data confirm and support the hypothesis that TGF-beta 1 is angiogenic in vitro, eliciting microvascular endothelial cells to form tube-like structures with apparent tight junctions and abluminal basal lamina deposition in three-dimensional cultures.

INFstrate-associated macromolecules promote cytodifferentiation of BC3H1 myogenic cells

Article

Mar 1991

Differentiated mouse BC3H1 myogenic cells secrete substrate-associated macro-molecules (SAM) which restrict the proliferation of undifferentiated cells and promote both cell shape changes and expression of predominantly the vascular smooth muscle (VSM)-specific isoform of the contractile protein alpha-actin. While we previously reported that high cell density was required for stimulating maximal expression of VSM alpha-actin in BC3H1 cells (Strauch and Reeser: Journal of Biological Chemistry 264:8345-8355, 1989), the permissive effect of SAM on myoblast cytodifferentiation was not at all dependent on the formation of cell to cell contacts. This observation suggests that biogenesis of an extracellular matrix rather than the formation of physical contacts between cells may be the rate-limiting step for induction of VSM alpha-actin expression at high cell density. The biologically active moieties in SAM that promote cytodifferentiation also are expressed by mouse embryonic fibroblast cell lines and are distinctly different from a class of adheron-like macromolecules released by differentiated BC3H1 myocytes directly into the culture medium. While SAM was cell growth restrictive, reconstituted particulate material (RPM) prepared from myocyte-conditioned medium promoted the adhesion and proliferation of growth-arrested myoblasts. SAM and RPM are composed of different polypeptide subunits which collectively may establish microenvironmental conditions that are permissive for BC3H1 myogenic cell differentiation.

JE mRNA accumulates rapidly in aortic injury and in PDGF-stimulated vascular smooth muscle cells

Article

Mar 1992

The early response to vascular injury is characterized by migration of inflammatory cells, including monocytes, and platelets to the damaged vessel wall. These inflammatory cells may serve as a source of growth factors and cytokines that stimulate vascular smooth muscle cell (VSMC) migration and proliferation associated with intimal hyperplasia. JE is a platelet-derived growth factor (PDGF)-inducible "early" gene that encodes a monocyte chemoattractant and, as such, could play an important role in inflammation. We now report that JE mRNA levels are increased in intact aorta after balloon injury. The time course of this increase, with maximal levels at 4 hours, is similar to that seen in PDGF-treated cultured rat aortic VSMCs. The accumulation of JE mRNA in cultured VSMCs is accompanied by a marked increase in the secretion of JE protein. The elevation of JE mRNA levels in VSMCs shows specificity for PDGF, because angiotensin II, alpha-thrombin, and epidermal growth factor fail to increase JE mRNA levels. In contrast to 3T3 fibroblasts, the accumulation of JE mRNA in VSMCs in response to PDGF is predominantly due to an increase in JE mRNA stability. The accumulation of JE mRNA in VSMCs stimulated by PDGF appears to occur via a novel pathway(s) independent of Ca2+ mobilization, Na(+)-H+ exchange, protein kinase C activation, or elevation in cAMP levels. These findings suggest that VSMCs may take part in the early inflammatory response after injury through the production of JE, a potent monocyte chemoattractant. Finally, our data suggest that JE may be a marker for PDGF-specific effects on VSMCs, both in vitro and in vivo. Thus, in addition to direct effects on VSMC growth and migration, PDGF may play a role in the early inflammatory response after vascular injury by inducing chemoattractants, such as that encoded by JE.

Cloning and characterization of synthetic sequences from the Xenopus laevis vitellogenin structural gene

Article

Full-text available

Jan 1979

The mRNA coding for vitellogenin, the yolk protein precursor, has been isolated from the liver of estrogen-stimulated Xenopus laevis. The mRNA has a size of 6.3 kilobases (kb). Optimal conditions were investigated for the synthesis of long complementary DNA (cDNA, referring to DNA synthesized in vitro) copies of the mRNA. Temperature, salt concentration, and enzyme-to-RNA ratio were important factors. Double-stranded cDNA with an average size of 2 to 3 kb was inserted into the vector pMB9 by the poly(dA:dT) method, and the recombinant plasmids were amplified in E. coli. Twenty-one clones with vitellogenin inserts ranging from 1 to 3.7 kb were studied. The regions in the RNA from which these clones had been derived were mapped by R-loop analysis in the electron microscope and by hybridization of the cloned DNAs with specific fractions of mRNA. Slightly more than half of the clones were derived from the 3′-terminal portions of the mRNA while the remaining clones are located internally.

High Resolution Two-Dimensional Electrophoresis of Proteins

Article

Full-text available

Jun 1975

Patrick H O'Farrell

A technique has been developed for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources. Proteins are separated according to isoelectric point by isoelectric focusing in the first dimension, and according to molecular weight by sodium dodecyl sulfate electrophoresis in the second dimension. Since these two parameters are unrelated, it is possible to obtain an almost uniform distribution of protein spots across a two-diminsional gel. This technique has resolved 1100 different components from Escherichia coli and should be capable of resolving a maximum of 5000 proteins. A protein containing as little as one disintegration per min of either 14C or 35S can be detected by autoradiography. A protein which constitutes 10 minus 4 to 10 minus 5% of the total protein can be detected and quantified by autoradiography. The reproducibility of the separation is sufficient to permit each spot on one separation to be matched with a spot on a different separation. This technique provides a method for estimation (at the described sensitivities) of the number of proteins made by any biological system. This system can resolve proteins differing in a single charge and consequently can be used in the analysis of in vivo modifications resulting in a change in charge. Proteins whose charge is changed by missense mutations can be identified. A detailed description of the methods as well as the characteristics of this system are presented.

Differential expression of gizzard actin genes during chick embryogenesis

Article

Full-text available

Dec 1979

The iso forms of gizzard actin present during chicken embryogenesis were analyzed by two-dimensional electrophoresis. During chick embryogenesis there are conspicuous changes in the content of the iso forms of actin. Gizzards from 8-day-old embryos contain almost exclusively beta-actin. After 8 days of embryonic age, there is a continuous increase in the amount of gamma-actin and an apparent decrease in the amount of beta-actin. These changes in the content of beta- and gamma-actin in gizzard tissue are paralleled by changes in the content of the corresponding mRNAs, as detected by the ability of the RNAs to direct the synthesis of these proteins in micrococcal nuclease-digested reticulocyte lysates. The correlation of the in vivo and in vitro experiments indicates that during chick embryogenesis there is a differential expression of the genes coding for the iso forms of gizzard actin and that this expression is controlled at the transcriptional level.

Morphologic and functional changes of the aortic intima during experimental hypertension

Article

Full-text available

Sep 1979

The morphology and permeability to horseradish peroxidase of the rat aortic intima have been investigated in three experimental models of hypertension having different values of plasma renin content and plasma aldosterone level. During hypertension the aortic endothelium shows three main changes: 1) increased arithmetic mean thickness, with prominent rough endoplasmic reticulum and polyribosomes; 2) the appearance of actin microfilament bundles; and 3) increased permeability to horseradish peroxidase. These changes are not present in all models, do not appear to depend on hypertension per se, and are independent of each other. The subendothelial layer of hypertensive animals shows an increased thickness that appears to be correlated with an increase of endothelial cell volume. Our results suggest that: 1) the aortic intima reacts differently to different types of hypertension, and 2) factors other than hypertension per se play a role in the development of vascular changes observed in animals with elevated blood pressure.

Sequential expression of actin genes during myogenesis

Article

Full-text available

May 1986

Embryonic muscle development permits the study of contractile protein gene regulation during cellular differentiation. To distinguish the appearance of particular actin mRNAs during chicken myogenesis, we have constructed DNA probes from the transcribed 3' noncoding region of the single-copy alpha-skeletal, alpha-cardiac, and beta-cytoplasmic actin genes. Hybridization experiments showed that at day 10 in ovo (stage 36), embryonic hindlimbs contain low levels of actin mRNA, predominantly consisting of the alpha-cardiac and beta-actin isotypes. However, by day 17 in ovo (stage 43), the amount of alpha-skeletal actin mRNA/microgram total RNA increased more than 30-fold and represented approximately 90% of the assayed actin mRNA. Concomitantly, alpha-cardiac and beta-actin mRNAs decreased by 30% and 70%, respectively, from the levels observed at day 10. In primary myoblast cultures, beta-actin mRNA increased sharply during the proliferative phase before fusion and steadily declined thereafter. alpha-Cardiac actin mRNA increased to levels 15-fold greater than alpha-skeletal actin mRNA in prefusion myoblasts (36 h), and remained at elevated levels. In contrast, the alpha-skeletal actin mRNA remained low until fusion had begun (48 h), increased 25-fold over the prefusion level by the completion of fusion, and then decreased at later times in culture. Thus, the sequential accumulation of sarcomeric alpha-actin mRNAs in culture mimics some of the events observed in embryonic limb development. However, maintenance of high levels of alpha-cardiac actin mRNA as well as the transient accumulation of appreciable alpha-skeletal actin mRNA suggests that myoblast cultures lack one or more essential components for phenotypic maturation.

Structure and complete nucleotide sequence of the chicken α-smooth muscle (aortic) actin gene. An actin gene which produces multiple messenger RNAs

Article

Full-text available

Aug 1986

The alpha-smooth muscle (aortic) actin gene is a distinct member of the actin multigene family which is expressed in vascular smooth muscle cells. We have determined the complete nucleotide sequence of 11 kilobase pairs of genomic DNA encoding the chicken alpha-smooth muscle actin gene. This single copy gene specifies a protein identical in sequence to the major alpha-actin from bovine aorta. The protein-coding sequences are interrupted by seven introns which are at codons specifying amino acid residues 41/42, 84/85, 121/122, 150, 204, 267, and 327/328. An eighth intron was found in the mRNA 5' untranslated region. The 5' flanking sequences contain elements which are conserved in other chicken muscle actin genes. Additional sequences at the 5' end of the gene may be conserved in at least one human actin gene. We have identified at least four messenger RNAs ranging in size from approximately 1370 to 2700 nucleotides (excluding poly(A) tails) which are transcribed from the alpha-smooth muscle actin gene. These RNAs differ in the length of their 3' untranslated regions, probably as a result of the utilization of alternative polyadenylation signals. This is the first report of an actin gene with multiple mRNA transcripts.

Cytoskeletal remodeling of rat aortic smooth muscle cells in vitro: Relationships to culture conditions and analogies to in vivo situations

Article

Full-text available

Aug 1986
J Submicr Cytol

Cytoskeletal features of arterial smooth muscle cells (SMC) vary characteristically during development and during atheromatous plaque formation (Gabbiani et al., 1984; Kocher et al., 1985). We have analyzed the cytoskeletal features of rat aortic SMC placed in culture in the presence of 10% foetal calf serum (thus containing growth factors probably playing a role in SMC development and atheroma formation), as compared to SMC freshly isolated from the rat aortic media. Under these conditions, SMC show a typical cytoskeletal remodeling characterized by: 1) increased content of vimentin per cell, increased number of cells containing only vimentin, and decreased number of vimentin plus desmin containing cells; 2) decreased contents of actin, tropomyosin and myosin; 3) a switch in the pattern of actin isoforms with the appearance of a beta-type predominance. Some of these changes (e.g. increase of vimentin and decrease of alpha-type actin) are seen already in cells entering for the first time in S-phase after plating. Pulse-chase experiments with 3H-thymidine (3H-TdR) indicate that vimentin containing SMC possess a higher replicative activity than vimentin plus desmin containing SMC, thus explaining the selection of vimentin containing cells during culture. Our results indicate that during culture SMC develop features similar to those observed in normal foetal SMC or in SMC present in atheromatous plaques; this model may be useful for the understanding of mechanisms leading to SMC differentiation and to atheroma formation.

Skalli, O, Ropraz, P, Trzeciak, A, Benzonana, G, Gillesen, D & Gabbiani, G. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 103: 2787-2796

Article

Full-text available

Jan 1987

A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha-smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha-smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti-desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin-negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.

Actin isoform synthesis and mRNA levels in quiescent and proliferating rat aortic smooth muscle cells in vivo and in vitro

Article

Full-text available

Sep 1986

The relative levels of actin isoform synthesis in rat aortic smooth muscle cells (SMC) have been studied in vivo and in culture by means of [35S]methionine incorporation. They have been compared to the functional levels of actin isoform mRNAs, assayed by translation of total cell RNA in a reticulocyte lysate or, in some cases, to the actin isoform RNA content, assayed by Northern-blot hybridization to a total actin cRNA probe. In normal media and in freshly isolated SMC, the relative levels of actin isoform synthesis and the actin mRNA translation products show a remarkable similarity, but differ from the proportions of actin isoforms present in a total cell extract (Gabbiani G, Kocher O, Bloom WS, Vandekerckhove J, Weber K: Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media. J Clin Invest 73:148, 1984); (Skalli O, Bloom WS, Ropraz P, Azzarone B, Gabbiani G: Cytoskeletal remodeling of rat aortic smooth muscle cells in vitro: relationship to culture conditions and analogies to in vivo situations. J Submicrosc Cytol, in press 1986). This suggests that different actin isoforms have different stabilities. Fifteen days after balloon induced endothelial denudation in vivo, and after being placed in culture, SMC show a decrease in the proportions of alpha-actin synthesis and alpha-actin mRNA levels with a corresponding increase in these parameters for beta- and lambda-actins. The proportions of actin isoform synthesis and actin mRNA translation products in intimal SMC revert to normal values 60 days after balloon induced endothelial denudation, when the aorta is reendothelialized; however, in culture decreased alpha-actin synthesis and mRNA level persist up to the fifth passage (P5). These changes may be helpful for the understanding of SMC adaptation mechanisms during arterial development and atheromatous plaque formation.

Development changes in isoactin expression in rat aortic smooth muscle cells in vivo. Relationship between growth and cytodifferentiation

Article

Full-text available

Nov 1986

There is an inverse relationship between cellular proliferation and smooth muscle alpha-isoactin expression in cultured vascular smooth muscle cells (SMCs) (Owens, G.K., Loeb, A., Gordon, D., and Thompson, M.M. (1986) J. Cell Biol. 102, 343-352). In the present studies, changes in isoactin expression were studied during developmental growth of rat aortic SMCs (ages 1-180 days) to better understand interrelationships between growth and cytodifferentiation in these cells in vivo. Actin expression (i.e. content and synthesis) was evaluated by one- and two-dimensional gel electrophoresis and using isoactin-specific antibodies. The major actin present in cells from newborn rats was nonmuscle beta-actin (56% of total actin), whereas cells from adult animals contained principally smooth muscle alpha-actin (Sm-alpha-actin) (76% of total actin). Increases in Sm-alpha-actin content with increasing age were due, in part, to an increase in Sm-alpha-actin synthesis. However, in SMCs from 90- and 180-day-old rats, the fractional content of Sm-alpha-actin exceeded its fractional synthesis at a time when total Sm-alpha-actin content was increasing. This suggests that Sm-alpha-actin turns over more slowly in mature animals. Decreases in the frequency of SMCs undergoing DNA synthesis with age could not account for increases in Sm-alpha-actin expression with age. However, combined immunocytological and [3H]thymidine autoradiographic studies demonstrated that nearly 50% of the medial derived cells from newborn rat aortas did not show detectable staining with a monoclonal antibody to smooth muscle-specific isoactins, and the replicative frequency was much higher in these cells than in cells that contained Sm-alpha-isoactins. Taken together, the results of the present studies and previous studies in cultured SMCs support the hypothesis that cessation of proliferation during development is associated with the induction of Sm-alpha-actin expression, but that factors other than cellular growth state play an important role in determining the level of Sm-alpha-actin expression in fully differentiated SMCs.

Characterization of actin mRNA levels in BC3H1 cell differentiation

Article

Full-text available

Feb 1986

The expression of a vascular smooth muscle specific alpha-actin isoform can be induced in mouse BC3H1 smooth muscle cells by treating confluent monolayers with serum-free medium (Strauch, A. R., and Rubenstein, P. A. (1984) J. Biol. Chem. 259, 3152-3159; 7224-7229). Using blot hybridization techniques, two size classes of actin RNA were identified in BC3H1 cells with the relative amount of RNA in each size class varying according to the developmental state of the cells; a 2100-nucleotide actin RNA was most abundant in myoblasts, whereas a smaller 1500-nucleotide actin RNA was found predominantly in fully differentiated myocytes. Results of in vitro translation experiments suggested that the 2100-nucleotide actin RNA on blots of myoblast total RNA corresponded to a mixture of similar size transcripts encoding both beta- and gamma-actin, while the 1500-nucleotide actin RNA in myocytes was an alpha-actin mRNA. Cell-cell contact and serum withdrawal initiated a 6-fold increase in the level of alpha-actin mRNA in BC3H1 cells that was followed by a 3-fold decrease in the amount of beta- and gamma-actin mRNA when confluent cells were exposed to serum-free medium for prolonged periods. Vascular smooth muscle alpha-actin was the major alpha-actin isoform synthesized in L-[35S]cysteine-labeled BC3H1 myocytes, indicating that the 1500-nucleotide actin mRNA size class in these cells may be enriched for vascular smooth muscle alpha-actin transcripts.

Expression of smooth muscle-specific α-isoactin in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation

Article

Full-text available

Mar 1986

The relationship between growth and cytodifferentiation was studied in cultured rat aortic smooth muscle cells (SMCs) using expression of the smooth muscle (SM)-specific isoactins (Vanderkerckhove, J., and K. Weber, 1979, Differentiation, 14:123-133) as a marker for differentiation in these cells. Isoactin expression was evaluated by: (a) measurements of fractional isoactin content and synthesis ([35S]methionine incorporation) by densitometric evaluation of two-dimensional isoelectric focusing sodium dodecyl sulfate gels, and (b) immunocytological examination using SM-specific isoactin antibodies. Results showed the following: (a) Loss of alpha-SM isoactin was not a prerequisite for initiation of cellular proliferation in primary cultures of rat aortic SMCs. (b) alpha-SM isoactin synthesis and content were low in subconfluent log phase growth cells but increased nearly threefold in density-arrested postconfluent cells. Conversely, beta-nonmuscle actin synthesis and content were higher in rapidly dividing subconfluent cultures than in quiescent postconfluent cultures. These changes were observed in primary and subpassaged cultures. (c) alpha-SM actin synthesis was increased by growth arrest of sparse cultures in serum-free medium (SFM; Libby, P., and K. V. O'Brien, 1983, J. Cell. Physiol., 115:217-223) but reached levels equivalent to density-arrested cells only after extended periods in SFM (i.e., greater than 5 d). (d) SFM did not further augment alpha-SM actin synthesis in postconfluent SMC cultures. (e) Serum stimulation of cells that had been growth-arrested in SFM resulted in a dramatic decrease in alpha-SM actin synthesis that preceded the onset of cellular proliferation. These findings demonstrate that cultured vascular SMCs undergo differential expression of isoactins in relation to their growth state and indicate that growth arrest promotes cytodifferentiation in these cells.

Efficient Invitro Synthesis of Biologically-Active Rna and Rna Hybridization Probes from Plasmids Containing a Bacteriophage-Sp6 Promoter.

Article

Full-text available

Oct 1984

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Structure of a human smooth muscle actin gene with a unique intron site

Article

Full-text available

Jul 1984

A recombinant phage containing an actin gene (lambda Ha201) was isolated from a human DNA library and the structure of the actin gene was determined. The amino acid sequences deduced from the nucleotide sequences of lambda Ha201 were compared with those of six actin isoforms; they matched those of bovine aortic smooth muscle actin, except for codon 309, which was valine (GTC) in lambda Ha201 and alanine (GCN) in bovine aortic smooth muscle actin. Southern blot hybridization experiments showed that the gene of normal human cells did not have the TaqI-sensitive site around position 309, whereas half of the genes of HUT14 cells did. These results indicate that one allele of the aortic smooth muscle actin gene in HUT14 cells has a transition point mutation (C----T) at codon 309 and that the amino acid sequences of normal human aorta and bovine smooth muscle actins are probably identical. In addition to the five introns interrupting exons at codons 150, 204, and 267, and between codons 41 and 42 and 327 and 328, which are common to skeletal muscle and cardiac muscle actin genes, the smooth muscle actin gene has two more intron sites between codons 84 and 85 and 121 and 122. The previously unreported intron site between codons 84 and 85 is unique to the smooth muscle actin gene. The intron site between codons 121 and 122 is common to beta-actin genes but is not found in other muscle actin genes. A hypothesis is proposed for the evolutionary pathway of the actin gene family.

Human actin genes are single copy for α-skeletal and α-cardiac actin but multicopy for β- and γ-cytoskeletal genes: 3' Untranslated regions are isotype specific but are conserved in evolution

Article

Full-text available

Nov 1983

We have constructed isotype-specific subclones from the 3' untranslated regions of alpha-skeletal, alpha-cardiac, beta-cytoskeletal, and gamma-cytoskeletal actin cDNAs. These clones have been used as hybridization probes to assay the number and organization of these actin isotypes in the human genome. Hybridization of these probes to human genomic actin clones (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981; Engel et al., Mol. Cell. Biol. 2:674-684, 1982) has allowed the unambiguous assignment of the genomic clones to isotypically defined actin subfamilies. In addition, only one isotype-specific probe hybridizes to each actin-containing gene, with a single exception. This result suggests that the multiple actin genes in the human genome are not closely linked. Genomic DNA blots probed with these subclones under stringent conditions demonstrate that the alpha-skeletal and alpha-cardiac muscle actin genes are single copy, whereas the cytoskeletal actins, beta and gamma, are present in multiple copies in the human genome. Most of the actin genes of other mammals are cytoplasmic as well. These observations have important implications for the evolution of multigene families.

Actin expression in smooth muscle cells of rat aortie intimal thickening and human atheromatous plaque

Article

Full-text available

Feb 1984

Actin of smooth muscle cells of rat and human aortic media shows a predominance of the alpha-isoform. In experimental rat aortic intimal thickening, in human atheromatous plaque, and in cultured aortic smooth muscle cells, there is a typical switch in actin expression with a predominance of the beta-form and a noticeable amount of gamma-form. This pattern of actin expression represents a new reliable protein-chemical marker of experimental and human atheromatous smooth muscle cells.

Induction of vascular smooth muscle alpha-isoactin expression in BC3HI cells

Article

Full-text available

Apr 1984

An isoactin analysis was performed on L-[35S]cysteine labeled BC3H1 cells to determine if these smooth muscle-like cells synthesize vascular smooth muscle actin. Three different NH2-terminal peptides were identified on thin layer electrophoretograms of DNase I-purified and trypsin-digested BC3H1 cell actin. Results obtained from secondary digestion with thermolysin or Staphylococcus aureus V8 protease showed that the most acidic NH2-terminal peptide was derived from vascular smooth muscle alpha-isoactin. Treatment of cell monolayers with serum-free medium caused a 3-fold increase in the level of alpha-isoactin expression and a concomitant decrease in the level of non-muscle beta- and gamma-isoactin. Cell-cell contact was required for induction of alpha-isoactin, and the effects of serum depletion on isoactin expression and cell growth were reversible. The intensity of about 11 out of 500 polypeptide spots on two-dimensional gels of BC3H1 cell polypeptides also was influenced by the culture conditions. The finding that smooth muscle isoactin expression was coupled to cell growth conditions indicate the potential usefulness of BC3H1 cells in studies of isoactin expression and utilization during vascular smooth muscle development.

A vascular smooth muscle alpha-isoactin biosynthetic intermediate in BC3H1 cells. Identification of acetylcysteine at the NH2 terminus

Article

Full-text available

Jul 1984

A fully translated actin biosynthetic intermediate containing N-acetylcysteine at the NH2 terminus has been identified in homogenates of differentiated mouse BC3H1 cerebrovascular smooth muscle cells labeled with L-[35S]cysteine. Thermolysin digestion of the highly acidic NH2-terminal tryptic peptide of this intermediate and electrophoretic analysis of the resulting fragments indicated that the intermediate was a precursor of smooth muscle alpha- isoactin , the major isoactin species in vascular smooth muscle. Carboxypeptidase A digestion of the thermolysin cleavage product corresponding to the first eight amino acid residues of the NH2-terminal tryptic peptide demonstrated an acetylcysteine-glutamate residue at the NH2 terminus. These results imply that the gene for smooth muscle alpha- isoactin , like genes coding for skeletal and cardiac alpha- isoactins , contains a cysteine codon immediately following the initiator methionine codon. Both the methionine and cysteine residues must be removed from the NH2 terminus of the intermediate to yield the mature form of smooth muscle alpha- isoactin . The removal of the cysteine residue in vivo is not direct but apparently involves acetylation of the cysteine and subsequent post-translational cleavage of the resulting acetylcysteine. Such an acetylation-dependent pathway has been demonstrated for removal of cysteine from the NH2 terminus of Drosophila actin synthesized in a cell-free translation system ( Rubenstein , P. A., and Martin, D. J. (1983) J. Biol. Chem. 258, 11354-11360). In vivo pulse-chase experiments indicate that the smooth muscle alpha- isoactin intermediate in BC3H1 cells turns over much more slowly than nonmuscle actin intermediates previously identified in mouse L-cells.

Isolation and characterization of full-length cDNA clones for human α, β, and γ-actin mRNAs: Skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed

Article

Full-text available

Jun 1983

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.

A monoclonal antibody against alpha-smooth muscle actin: A new probe for smooth muscle differentiation

Article

Jan 1986

Expression of smooth muscle-specific alpha-isoactin in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation

Article

Feb 1986

G. K. Owens

Golgen Des Gafass Kaheterismus AM Normo Und Hypercholesterinaemischen Kaninchen

Article

Jan 1966
PATHOBIOLOGY

The smooth muscle in culture

Article

Feb 1979

The current state of knowledge of both vascular and visceral smooth muscle in cell and tissue culture and the variety of preparations used for different experimental purposes are described. Organ culture of smooth muscle tissues is referred to only periodically.

An improved method for detecting foreign DNA in plasmids of Escherichia coli

Article

Oct 1979

Ronald E. Thayer

A new procedure has been developed for lysing bacterial colonies on nitrocellulose filters and immobilizing the released DNA on the filters. The procedure involves the use of lysozyme and Triton X-100. When used in conjunction with in situ hybridization, this method has proven effective in detecting DNA recombinants, while eliminating the problems of false positives and variation between duplicate filters that are seen with other methods.

A Rapid Alkaline Extraction Prodedure for Screening Recombinant Plasmid DNA

Article

Dec 1979

A procedure for extracting plasmid DNA from bacterial cells 1s described. The method 1s simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.

The Complete Amino Acid Sequence of Actins from Bovine Aorta, Bovine Heart, Bovine Fast Skeletal Muscle, and Rabbit, Slow Skeletal Muscle

Article

Feb 1979

Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal. The two smooth muscle actins—bovine aorta actin and chicken gizzard actin—differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared. In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably closer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.

Isolation of biologically active RNA from sources enriched in RNAse

Article

Dec 1979

Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.

At least six different actins are expressed in a higher mammal: An analysis based on the amino acid sequence of the amino-terminal tryptic peptide*1

Article

Jan 1979

The protein chemical characterization of the amino-terminal tryptic peptide of actin from different bovine tissues shows that at least six different actin structural genes are expressed in this mammal.Unique amirio acid sequences are found for actin from skeletal muscle, for actin from heart muscle, for two different actin species from smooth muscle, and for two different actin species typical of non-muscle tissues such as brain and thymus. The presence of more than one actin species in the same tissue (e.g. nonmuscle tissues and smooth muscles) is demonstrated by different amino-terminal peptides which, however, are closely related. The actins from the sarcomeric muscles (e.g. skeletal muscle and heart muscle) show unique but extremely similar amino-terminal peptides. A limited comparison of bovine and avian actins involving smooth and skeletal muscles emphasizes that among higher vertebrates actin divergence involves tissue rather than species specificity.For the lower eukaryotic organism Physarum polycephalum a single actin amino-terminal peptide is found, indicating that only one actin species is present during the plasmodial stage. The amino acid sequence of this peptide although unique reveals a high degree of homology with the corresponding mammalian cytoplasmic actin peptides.Different actin extraction and purification procedures have been compared by the relative yields of the different amino-terminal peptides. The results indicate that the various actin species obtained by the current purification procedures are a true reflection of the actual actins present in the tissue. In addition we compare the resolution provided by either isoelectric focusing analysis of different actins or by the protein chemical characterization of the amino-terminal peptides of different actins. We show that the latter procedure is more suitable for recording changes in actin expression during evolution and differentiation.

Protein synthesis and actin heterogeneity in calf muscle cells in culture Proc

Article

Jul 1976

Pulse-labeled cytoplasmic proteins from cultured fetal calf-muscle cells at various stages of development were analyzed by one- and two-dimensional gel electrophoresis. The high resolution of the two-dimensional technique allows the determination of those protein species that begin to be synthesized after cell fusion. In addition, actin has been found to exist in three forms possessing similar biochemical properties and identical molecular weights but having slightly different isoelectric points. Two of the forms are found in prefusion dividing myoblasts and also in cultured kidney cells. The third form is the only one found in fetal muscle tissue and is predominant in cultures of fused muscle cells. Thus, it would seem that actin can exist in several isozymic forms of which one is specific to fused muscle tissue.

Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis

Article

Dec 1975

Ed Southern

This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.

Colony Hybridization: A Method for the Isolation of Cloned DNAs that Contain a Specific Gene

Article

Feb 1992
Biotechnology

A method has been developed whereby a very large number of colonies of Escherichia coli carrying different hybrid plasmids can be rapidly screened to determine which hybrid plasmids contain a specified DNA sequence or genes. The colonies to be screened are formed on nitrocellulose filters, and, after a reference set of these colonies has been prepared by replica plating, are lysed and their DNA is denatured and fixed to the filter in situ. The resulting DNA-prints of the colonies are then hybridized to a radioactive RNA that defines the sequence or gene of interest, and the result of this hybridization is assayed by autoradiography. Colonies whose DNA-prints exhibit hybridization can then be picked from the reference plate. We have used this method to isolate clones of ColE1 hybrid plasmids that contain Drosophila melanogaster genes for 18 and 28S rRNAs. In principle, the method can be used to isolate any gene whose base sequence is represented in an available RNA.

Actin-isoform pattern as a marker of normal or pathological smooth-muscle and fibroblastic tissues

Article

Feb 1987

The relative proportions of actin isoforms present in smooth-muscle (SM) and fibroblastic human and non-human tissue extracts were examined by densitometric evaluation of Coomassie-Blue-stained spots in two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as well as by quantification of radiolabeled actin NH2-terminal peptide spots separated by two-dimensional paper electrophoresis. SM tissues contained alpha- and gamma-SM as well as beta- and gamma-cytoplasmic (CY) actins in different proportions in different organs. Species differences with respect to the ratios of the isoactins were also observed. Moreover, during pregnancy, both human and rat myometrium exhibited a changed actin-isoform pattern, there being an increased proportion of gamma-actin. Analysis of the NH2-terminal peptides showed that, in human myometrium, this was essentially due to an increase in the amount of the gamma-SM isoform. Fibroblastic tissues were found to contain only the beta- and gamma-CY isoforms, the ratio being approximately 2.6:1. Thus, the presence or absence of alpha-actin provides a reliable biochemical criterion for distinguishing between fibroblastic and SM cell populations and/or tissues. This distinction and the evaluation of changes in isoactin ratios may be useful in the study of differentiation as well as physiological and pathological phenomena, and for determining the origin of certain soft-tissue tumours.

Cytoskeletal features and atheromatous human arterial smooth muscle cells

Article

Oct 1986
Hum Pathol

The distribution of actin, vimentin, desmin, and tropomyosin was studied in the media of the human aorta and femoral and coronary arteries, as well as in atheromatous plaques from the same arteries, by means of immunofluorescence, densitometric analysis of sodium dodecylsulfate-polyacrylamide gel electrophoresis, and bidimensional gel electrophoresis. The proportions of desmin-containing cells varied in the media of different arteries; 4 per cent of the cells in the aorta, 11 per cent in the coronary artery, and 37 per cent in the femoral artery contained desmin. In fibrous atheromatous plaques, independently of the artery, desmin-containing cells were almost absent, but they reappeared in complicated lesions. The content of vimentin per smooth muscle cell increased in fibrous atheromatous plaques, whereas the content of actin and tropomyosin was less than in normal media. Moreover, the alpha-actin predominance observed in the media was transformed to beta-actin predominance in the atheromatous plaques. These cytoskeletal changes provide new, possibly useful, biochemical markers for the characterization of smooth muscle cells during early and advanced phases of atheroma formation.

Expression of actin mRNAs in rat aortic smooth muscle cells during development, experimental intimal thickening, and culture

Article

Feb 1986

The expression of actin-isoform mRNAs in the smooth muscle cells (SMC) of the aortic media in rats has been studied by Northern-blot hybridization, using a general actin-cRNA probe, and two cRNA probes specific for beta- and gamma-cytoplasmic actins, during: (1) development, (2) intimal thickening after endothelial injury induced by balloon catheterization, and (3) growth in culture. In 5-day-old rats, the ratio between alpha-smooth-muscle-actin mRNA and beta- and gamma-cytoplasmic-actin mRNAs was close to 1. It increased to about 4 in 6-week-old rats. Replicating SMC from regions of intimal thickening 15 days after endothelial injury, and SMC growing in culture contained a predominance of cytoplasmic actin mRNAs. Intimal SMC 60 days after endothelial injury (at which time the endothelium had fully regenerated) demonstrated a pattern of actin mRNAs similar to that of normal media. Functional mRNA measured by translation in a reticulocyte lysate showed increases in the level of alpha-actin and decreases in beta-actin in rats from 5 days to 6 weeks of age. These results suggest that during development, under pathological conditions, and in cell culture, the expression of actin isoforms in arterial SMC depends on many factors, including the amount and translation efficiency of mRNAs, and the relative stabilities of the proteins involved.

Cytoskeletal features of rat aortic cells during development. An electron microscopic, immunohistochemical, and biochemical study

Article

Jul 1985

Actin, vimentin, desmin, and tropomyosin distribution in rat aortic endothelial and smooth muscle cells has been studied during development using fetal (18 to 20 days of gestation), and 5- and 14-day-, and 5-, and 12-week-old rats. Endothelial cells of newborn animals actively replicate and contain many actin stress fibers, whereas, in adult animals, replication is minimal and actin stress fibers are rare. The actin, vimentin, desmin, and tropomyosin content of smooth muscle cells increases gradually from fetal to adult animals. The number of desmin-containing cells also increases from 13% in fetal rats to 51% in adult rats. The beta-actin isoform is predominant in fetal and newborn animals, but gradually the alpha-isoform becomes quantitatively the most important, as seen by bidimensional polyacrylamide gels. Several analogies exist between the features of developing smooth muscle and what is known for developing striated muscle cells. The evolution of cytoskeletal features from fetal to adult animals is remarkably the opposite of what takes place in: (1) rat aortic smooth muscle cells proliferating after an endothelial injury, (2) human arterial smooth muscle cells present in atheromas, and (3) actively growing rat aortic smooth muscle cells in vitro. Thus, the assumption that pathological or cultured smooth muscle cells are "dedifferentiated" is supported by our biochemical observations.

Smooth muscle α-actin is a marker for mouse NIH 3T3 and Rat-2 cells

Article

Aug 1985

Heteroploid mouse NIH 3T3 fibroblasts and several rat fibroblast strains (Rat-1, Rat-2 and REF-52) are cell lines of special interest in the field of carcinogenesis because of their extensive use as normal cells in transformation assays for putative cancer-causing genes. Exposure of these cells to carcinogenic chemicals or oncogenic DNA produces anchorage-independent cells with retracted cytoplasms that lack actin cables. All human fibroblast strains, normal and transformed, synthesize two electrophoretic forms of actin (beta- and gamma-actin). In contrast, we discovered that early-passage mouse and rat strains synthesize abundant amounts of each of the three electrophoretic forms of actin (alpha-, beta- and gamma-actin) but mouse and rat cancer cells express only beta- and gamma-actins. We now show that in NIH 3T3 and Rat-2 fibroblasts a third actin, the smooth muscle alpha isoform, is abundantly co-expressed with beta- and gamma-actin. In every instance tested following transformation to tumorigenicity, the accumulation of alpha-actin messenger RNA and alpha-actin synthesis was greatly inhibited. Shutdown of alpha-actin expression thus appears to be a reproducible transformation-sensitive marker in rodent fibroblasts.

Kinetics of cellular proliferation after arterial injury. II. Inhibition of smooth muscle growth by heparin

Article

Jul 1985

Heparin inhibits intimal thickening after arterial injury. Whether this effect is due to inhibition of medial smooth muscle cell (SMC) migration, SMC proliferation in the intima, or synthesis and deposition of connective tissue has not been evident. In this study we have investigated these possibilities in a rat carotid balloon injury model. Heparin (0.3 mg/kg/hour) was administered intravenously by means of osmotic pumps to experimental animals, and controls received lactated Ringer's solution. Smooth muscle proliferation (thymidine index), intimal smooth muscle accumulation, and endothelial regeneration were measured at intervals between 0 and 28 days. Total smooth muscle growth as determined biochemically at 14 days was markedly inhibited by heparin if the pumps were placed 24 hours before or at the time of injury and less so if inserted 48 or 96 hours after injury. SMC thymidine indices were maximal in the media at 4 days and in the intima at 7 days for injured arteries of both heparin-treated and control rats; at each time point SMC proliferation and intimal thickening were less in heparin-treated rats. The volume of connective tissue in the intima was the same in both groups at 28 days. Medial SMC migration into the intima was diminished by heparin treatment, but endothelial regeneration was not affected. These results support the hypothesis that heparin is a specific inhibitor of SMC migration and proliferation and is most effective if started before SMC enter S-phase.

Purification of Biologically Active Globin Messenger RNA by Chromatography on Oligothymidylic Acid-Cellulose

Article

Jul 1972

A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described. The method depends upon annealing poly(adenylic acid)-rich mRNA to oligothymidylic acid-cellulose columns and its elution with buffers of low ionic strength. Biologically active rabbit globin mRNA has been purified by this procedure and assayed for its ability to direct the synthesis of rabbit globin in a cell-free extract of ascites tumor. Inasmuch as various mammalian mRNAs appear to be rich in poly(adenylic acid) and can likely be translated in the ascites cell-free extract, this approach should prove generally useful as an initial step in the isolation of specific mRNAs.

Effects of vascular catheterization in normo- and hypercholesteremic rabbits

Article

Feb 1966

Maxam, A.M. & Gilbert, W. Sequencing end-labeled DNA with base specific chemical cleavages. Meth. Enzymol. 65, 499-560

Article

Feb 1980
METHOD ENZYMOL

This chapter discusses the sequencing end-labeled DNA with base-specific chemical cleavages. In the chemical DNA sequencing method, one end-labels the DNA, partially cleaves it at each of the four bases in four reactions, orders the products by size on a slab gel, and then reads the sequence from an autoradiogram by noting which base-specific agent cleaved at each successive nucleotide along the strand. This technique sequences the DNA made in and purified from cells. No enzymatic copying in vitro is required, and either single- or double-stranded DNA can be sequenced. Most chemical schemes that cleave at one or two of the four bases involve three consecutive steps: modification of a base, removal of the modified base from its sugar, and DNA strand scission at that sugar. Base-specific chemical cleavage is only one step in sequencing DNA. The chapter presents techniques for producing discrete DNA fragments, end-labeling DNA, segregating end-labeled fragments, extracting DNA from gels, and the protocols for partially cleaving it at specific bases using the chemical reactions. The chapter also discusses the electrophoresis of the chemical cleavage products on long-distance sequencing gels and a guide for troubleshooting problems in sequencing patterns.

A reliable method for the recovery of DNA fragments from agarose and acrylamide gels

Article

May 1981

We describe a simple and efficient method for the recovery of DNA fragments from agarose or acrylamide gels. The procedure involves electrophoresis of the fragments onto strips of DEAE-cellulose paper inserted in the gel between bands visualized by ethidium bromide fluorescence. The electrophoretic transfer is achieved in the original resolving gel using the same apparatus, thereby enabling purification of DNA fragments up to 20 kb in size with a yield of 60–80%. DNA which has been recovered in this way can be used for restriction enzyme cleavage, nick translation, end labeling by T4 polynucleotide kinase, DNA sequencing, and electron microscopy. The DNA fragments can be recloned in pBR322 using either blunt-end or cohesive-end ligation. Thus our method is useful for the recovery of biologically active DNA from both agarose and high-percentage polyacrylamide gels.

An improved procedure or utilizing terminal transferase to add homopolymers to the 3′ termini of DNA

Article

Sep 1981

Terminal deoxynucleotidyl transferase (E.C.2.7.7.3.1.) from calf thymus was used to add homopolymer tails to duplex DNA with 3′ protruding, even, or 3′ recessive ends. A gel electrophoresis method was employed to analyze the tail length and the percent of DNA with tails. In all the tailing reactions, dA, dT, and dC tails from CoCl2-containing buffer were longer than those from MnCl2– or MgCl2–-containing buffers, whereas dG tails from MnCl2–containing buffer were the longest. By varying the ratio of dNTP over DNA terminus and the concentration of terminal transferase, optimal conditions were found for adding dG or dC tails of 10–25 nucleotides in length and dA and dT tails of 20–40 nucleotides in length to duplex DNA with all types of 3′ termini.

The role of Protein breakdown in growth, quiescence, and starvation of vascular smooth muscle cells

Article

Mar 1984

Protein accumulation in growing cells may be due in part to a reduction in the rate of protein breakdown. Previous studies of the relation of cell proliferation to protein degradation often produced growth arrest by conditions that may involve nutritional deprivation. However, nutrient lack can itself accelerate proteolysis and produce negative protein balance. We therefore reexamined the relation between growth and protein breakdown using a more selective method for limiting cell growth. We produced quiescent cell cultures using a chemically defined, serum-free medium supplemented with hormones and nutrients. Such media can maintain viability and near neutral protein balance in cultured vascular smooth muscle cells, in part because of reduced breakdown of cellular protein. We then compared rates of protein degradation in these quiescent but not starving cells, to those of cultures stimulated to grow by addition of mitogenic substances. Platelet-derived growth factor, fibroblast growth factor, or fetuin added to insulin-containing medium stimulated growth of smooth muscle cells, but further reduced protein breakdown only slightly. Contrary to the implications of certain previous studies, our results show that proliferating cells can accumulate protein without an appreciable reduction in the rates of protein breakdown. Thus, while accelerated proteolysis appears to be an important adaptation to adverse nutritional conditions, growth of smooth muscle cells does not require changes in overall protein breakdown, but occurs primarily through an increase in protein synthesis.

Structure of two human ??-actin-related processed genes one of which is located next to a simple repetitive sequence

Article

Feb 1983

From a human gene library we have isolated and sequenced a beta-actin-like pseudogene, H beta Ac-psi 2, which lacks intervening sequences and contains several mutations resulting in frame-shifts, stop codons and in a departure from the known beta-actin protein sequence. We have also extended our sequence work on the intronless human beta-actin-related pseudogene H beta Ac-psi 1 described previously and we find that both genes are processed genes ending in a poly(dA) tract and flanked by direct repeats. The gene H beta Ac-psi 2 is preceded by a 230-bp region in which the simple sequence 5'-GAAA-3' is repeated greater than 40 times. This satellite-like sequence is highly repetitive in the human genome.

Phenotype modulation in primary cultures of arterial smooth muscle cells. On the role of platelet-derived growth factor

Article

Feb 1983

Smooth muscle cells were isolated from adult rat aorta by collagenase digestion, grown in primary culture in the presence of 10% whole blood serum (WBS), and studied by quantitative electron microscopy and thymidine autoradiography in order to correlate cellular fine structure and proliferation. On day 2–4, the cells passed through a structural transition from contractile to synthetic state. In the former they were characterized by predominance of cytoplasmic microfilament bundles and in the latter by an extensive rough endoplasmic reticulum (RER) and a large Golgi complex. The disappearance of the microfilament bundles was accompanied by a transient increase in lysosomal volume density but no signs of bulk autophagy. This suggests that microfilaments were disassembled into subunit proteins and that lysosomes were engaged in adjusting the pool of free subunits into a new equilibrium. RER. cisternae grew out from the nuclear envelope and successively spread throughout the cytoplasm. Stacks of Golgi cisternae were organized in a circumscribed juxtanuclear region. The structural modulation occurred also in medium containing 10% plasma-derived serum (PDS). Its onset was delayed by addition of antibodies (50 μg/ml) against platelet-derived growth factor (PDGF) to 10% WBS-medium and speeded up by addition of purified PDGF (25 ng/ml) to 10% PDS-medium. Otherwise, the kinetics of the structural modulation was the same in all experimental groups. The observations could not be explained by overgrowth of contaminating fibroblasts since (1) successive steps in the process were clearly evident, (2) the cells surrounded themselves by an incomplete basement membrane, a characteristic feature of smooth muscle, and (3) mitomycin C blocked cell growth but not conversion from contractile to synthetic state.

Human atherosclerosis. I. Cell constitution and characteristics of advanced lesions of the superficial femoral artery

Article

Feb 1984

This study represents a systematic analysis of the fine-structural characteristics of atherosclerotic lesions of the superficial femoral artery in man together with the growth characteristics in culture of the smooth muscle cells derived from these lesions. Occlusive fibrous atherosclerotic plaques were obtained from 29 male patients at the time of bypass surgery for occlusion of the superficial femoral artery and were studied by light and transmission electron microscopy. The occluded segment of each artery was obtained immediately after removal from the patient and examined with sterile techniques, and representative segments were fixed for light- and electron-microscopic study. Adjacent segments were used for dissection of the lesion away from the underlying media, and smooth muscle cells were cultured from lesion and nonlesion areas and compared in terms of their growth responses to increasing concentrations of a pool of human whole blood serum. The majority of the lesions were fibroproliferative and contained relatively little lipid. The fibrous cap that covered each lesion consisted of a special form of dense connective tissue that contained flat, pancake-shaped smooth muscle cells in a lacunalike space. This space consisted of concentric layers of basement membrane, collagen fibrils, and proteoglycan. The majority of the cells beneath the fibrous cap were smooth muscle cells mixed with small but varying numbers of macrophages. Most of the lesions were occluded by a thrombus, which had undergone organization and recanalization. A small number of the lesions had deep lipid deposits together with foci of degeneration and calcification. The occluded thrombi contained smooth muscle cells and a larger proportion of macrophages than the lesions themselves. The in vitro growth properties of the smooth muscle cells isolated from the lesion and the underlying media suggested that the lesion cells had senesced, compared with the medial smooth muscle cells derived from the same artery.

Cytoskeleton of rat aortic smooth muscle cells: Normal conditions and experimental intimal thickening

Article

Jul 1984

The organization of actin, vimentin, and desmin in smooth muscle cells of rat aortic media and intima under normal conditions and 15 or 75 days after endothelial injury has been studied by means of electron microscopy, indirect immunofluorescence, densitometric analysis of sodium dodecyl sulfate-polyacrylamide gels, isoelectric focusing, and bidimensional gels. In the normal aortic media, practically all smooth muscle cells contain vimentin, and about 50% of them contain, in addition, desmin; upon analysis of actin isotypes by bidimensional gels, smooth muscle cells show a predominance of alpha-actin, some beta-actin, and very little gamma-actin. Fifteen days after endothelial injury, cells that have migrated into the intima contain decreased amounts of actin and desmin and increased amounts of vimentin compared with normal medial smooth muscle cells. Moreover, beta-actin becomes the predominant actin isotype and significant amounts of gamma-actin appear, whereas alpha-actin decreases. Seventy-five days after endothelial injury, regenerated endothelial cells have repaired the injury. Intimal smooth muscle cells are less numerous than 15 days after injury, and the organization of their cytoskeletal elements has reverted almost to normal conditions. At both 15 and 75 days after endothelial injury, no significant changes of cytoskeletal elements are seen in the aortic media underlying intimal thickenings.

Changes in actin synthesis and α-actin-mRNA content in rat muscle during immobilization

Article

Aug 1984
Am J Physiol

The fractional rates of actin synthesis in adult rat gastrocnemius muscle from control and 6-h hindlimb-immobilized animals were determined by the constant-infusion technique. The rate of actin synthesis in gastrocnemius muscle was significantly decreased from control values during the 6th h of hindlimb immobilization. The content of alpha-actin-specific mRNA was then estimated in adult rat gastrocnemius muscle from control, 6-h, 72-h, and 7-day immobilized animals by "dot blot" hybridization. RNA extracted from control and immobilized animals was secured on nitrocellulose filters and hybridized to 32P-labeled plasmid p749 (containing a cDNA sequence produced from rat alpha-actin mRNA). The relative content of alpha-actin-specific mRNA in gastrocnemius muscle was significantly decreased at 7 days of immobilization but not at 6 or 72 h of immobilization. It is concluded that a change in the content of alpha-actin mRNA does not contribute significantly to the rapid onset of the decrease in actin synthesis rate observed after 6 h of immobilization. An alteration in the translation of alpha-actin-specific mRNA must occur to account for the early decline in actin synthesis during immobilization.

Coexistence of vimentin and desmin type intermediate filaments in a subpopulation of adult rat vascular smooth cells growing in primary culture

Article

Jun 1982

The differential expression of intermediate filaments in primary cultured adult rat vascular smooth muscle cells derived from the thoracic aorta was studied by double label immunofluorescence microscopy using antibodies to vimentin and desmin. Three distinct cell types are documented. Cells rich in vimentin and lacking desmin, cells expressing both vimentin and desmin and finally cells rich in desmin but lacking vimentin. The results confirm the previously proposed heterogeneity of the cellular populations of vascular smooth muscle in situ and prove the co-existence of desmin and vimentin in the same differentiated cell type obtained from an adult animal. The results also raise the possibility that BHK21 cells which express both desmin and vimentin in culture may have originated from vascular smooth muscle.

Remodeling of the rat aortic endothelial layer during experimental hypertension. Changes in replication rate, cell density, and surface morphology

Article

Feb 1983

Three models of hypertension were induced in Wistar rats: (1) aortic ligature between renal arteries, (2) uninephrectomy and Na-rich diet, (3) uninephrectomy, 0.9 per cent NaCl as drinking fluid and subcutaneous administration of desoxicorticosterone acetate (DOCA). We studied the aortic endothelium during the early (7 to 10 days) and late (40 days) phases of these models and quantified: (1) thymidine index and cell density using autoradiography on en face preparations, and (2) internal aortic circumference by planimetry. We also examined by means of scanning electron microscopy the surface morphology of the aorta in rats with aortic ligature. Thymidine index was constantly increased in the early phases of all models; it reverted to normal levels in the late phases. Endothelial cell density was slightly but significantly increased in the early and late phases after aortic ligature and in the early phase after Na-rich diet; it increased strikingly in the early and late phases after DOCA. There were no significant correlations between the variations of the internal aortic circumference and blood pressure, thymidine index, or cell density. Scanning electron microscopy showed no discontinuity of the endothelial cell layer either in the early or late phases after aortic ligature; bulging of endothelial cells into the lumen and appearance of cell clusters with numerous surface projections were seen during the early phase after aortic ligature. These changes produce a remodeling of the aortic endothelial layer which is maximal in the early phase after aortic ligature and in the early and late phases after DOCA and correlates with the previously reported increase of endothelial permeability to horseradish peroxidase in the same hypertensive situations.

Analysis of α-smooth muscle actin mRNA expression in rat aortic smooth muscle cells using a specific DNA probe

Abstract

No full-text available

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