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Cytoskeletal features of rat aortic cells during development. An electron microscopic, immunohistochemical, and biochemical study
Abstract
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.
... Thus, it appears that the myofibroblasts acquire many features of cultured fibroblasts . Up to now, the mammalian nonmuscle cells in which stress fibers have been noted are myofibroblasts (Gabbiani and Rungger-Brandle, 1981), endothelial cells of large vessels White et al., 1983;Wong et al., 1983;Kocher et al., 1985), perineural cells (Ross and Reith, 1969), brain pericytes (Le Beux and Willemot, 1978), and Sertoli cells (Toyama, 1976;Toyama et al., 1979). ...
... In vitro, cultured vascular smooth muscle cells develop characteristics corresponding to a morphologic "synthetic" phenotype, as compared to the "contractile" phenotype generally present in vivo (Chamley-Campbell et al., 1979). In vivo, smooth muscle cells with a phenotype similar to the in vitro "synthetic" phenotype were reported in the normal chicken aortic media (Moss and Benditt, 1970), in the human myometrium during pregnancy (Laguens and Lagrutta, 1964), in rat myometrium after estrogen treatment (Ross and Klebanoff, 1967), in the developing rat aortic media (Kocher et a]., 1985;Olivetti et al., 1980), and in rat aortic experimental thickening induced by endothelial injury Poole et al., 1971) (Fig. 8). ...
... Another important point is that fibroblasts and smooth muscle cells grown in culture resemble the myofibroblast counterpart in many respects; this has been shown for vascular smooth muscle cells Kocher et aL, 1985;Skalli et al., 1986b) and suggested for fibroblasts (Gabbiani and Rungger-Brandle, 1981;Gabbiani et aL, 1973;Guber and Rudolph, 1978) (see Section 2.1). It is tempting to speculate that when placing fibroblasts and smooth muscle cells in culture, one mimics a pathologic transition. ...
... Thus, it appears that the myofibroblasts acquire many features of cultured fibroblasts . Up to now, the mammalian nonmuscle cells in which stress fibers have been noted are myofibroblasts (Gabbiani and Rungger-Brandle, 1981), endothelial cells of large vessels White et al., 1983;Wong et al., 1983;Kocher et al., 1985), perineural cells (Ross and Reith, 1969), brain pericytes (Le Beux and Willemot, 1978), and Sertoli cells (Toyama, 1976;Toyama et al., 1979). ...
... In vitro, cultured vascular smooth muscle cells develop characteristics corresponding to a morphologic "synthetic" phenotype, as compared to the "contractile" phenotype generally present in vivo (Chamley-Campbell et al., 1979). In vivo, smooth muscle cells with a phenotype similar to the in vitro "synthetic" phenotype were reported in the normal chicken aortic media (Moss and Benditt, 1970), in the human myometrium during pregnancy (Laguens and Lagrutta, 1964), in rat myometrium after estrogen treatment (Ross and Klebanoff, 1967), in the developing rat aortic media (Kocher et a]., 1985;Olivetti et al., 1980), and in rat aortic experimental thickening induced by endothelial injury Poole et al., 1971) (Fig. 8). ...
... Another important point is that fibroblasts and smooth muscle cells grown in culture resemble the myofibroblast counterpart in many respects; this has been shown for vascular smooth muscle cells Kocher et aL, 1985;Skalli et al., 1986b) and suggested for fibroblasts (Gabbiani and Rungger-Brandle, 1981;Gabbiani et aL, 1973;Guber and Rudolph, 1978) (see Section 2.1). It is tempting to speculate that when placing fibroblasts and smooth muscle cells in culture, one mimics a pathologic transition. ...
... These tissue extracts and the products of in vivo synthesis, in vitro translation, or cell sorting, were stored at -20°C. For SDS-PAGE, these extracts were diluted 1:2 in sample buffer containing 1% SDS, 1% DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and I mM Na-P-Tosyl-L-arginine methylester in 0.0625 M Tris-HCI, pH 6.8, as previously described (23,24), and 40 p.g of protein were loaded on 10% gels with a 3% stacking gel under reducing conditions (24). For twodimensional gel electrophoresis, the extracts were diluted 1:5 with buffer A according to the method of O'Farrell (30), and between 20 and 50 p.g of protein were loaded. ...
... The second dimension was run on 10% polyacrylamide gels. For quantification, the gels of total carotid protein extract were stained with Coomassie Blue, and the relative proportions of actin isoforms were quantified by densitometry (23). Gels of products of in vivo synthesis or in vitro translation were dried and exposed to Kodak X-Omat SO-282 film. ...
... Gels of products of in vivo synthesis or in vitro translation were dried and exposed to Kodak X-Omat SO-282 film. The relative percentage of actin isoforms was determined by densitometry (1,23). ...
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.
... is another feature characterizing mature SMCs (19,20). Intermediate filaments in mature SMCs are composed of desrnin and vimentin whereas in immature SMCs the filaments are almost exclusively composed of vimentin (21). Organelles associated with protein synthesis (eg. ...
... Proliferatiog cells exhibit a reduction in smooth muscle or-and y-actuis (15), sm-MHC isoforms (22), smooth muscle regulatory 20KD MLC isoform (23) and h-caldesmon (26), the sarcoplasmic reticulurn membrane-associated protein, phospholamban (1 5). and the cytoskeletai proteins, desmin (21) and meta-vinculin (24). Furthermore, phenotypically modulated cultured cells are known to re-express foetal and non-muscle isoforms of several proteins including 0-actin (25), nrnMHC (22), the non-muscle regulatory myosin Iight chah isoform (23), f-caldesrnon (26), the intermediate filament associated protein, vimentin (21) and several secreted proteins including collagen and elastin (27). ...
... and the cytoskeletai proteins, desmin (21) and meta-vinculin (24). Furthermore, phenotypically modulated cultured cells are known to re-express foetal and non-muscle isoforms of several proteins including 0-actin (25), nrnMHC (22), the non-muscle regulatory myosin Iight chah isoform (23), f-caldesrnon (26), the intermediate filament associated protein, vimentin (21) and several secreted proteins including collagen and elastin (27). ...
... THE EARLY POSTNATAL PERIOD is associated with remarkable changes in the morphology and function of arterial endothelium. Endothelial cells lining fetal or newborn arteries are highly unusual, displaying prominent stress fibers (15,16,23) and reduced endothelial nitric oxide (NO)-mediated dilatation (1,3,8,27). Although common in cultured cells, arterial endothelial cells in vivo generally lack stress fibers, and F-actin is restricted to a faint cortical network at the cell periphery (7,10,15,16). ...
... Endothelial cells lining fetal or newborn arteries are highly unusual, displaying prominent stress fibers (15,16,23) and reduced endothelial nitric oxide (NO)-mediated dilatation (1,3,8,27). Although common in cultured cells, arterial endothelial cells in vivo generally lack stress fibers, and F-actin is restricted to a faint cortical network at the cell periphery (7,10,15,16). Indeed, as newborn arteries mature in the immediate postnatal period, the endothelial actin cytoskeleton remodels into a peripheral cortical network and there is a marked increase in endothelium-dependent dilatation (1,3,8,15,16,27). ...
... Although common in cultured cells, arterial endothelial cells in vivo generally lack stress fibers, and F-actin is restricted to a faint cortical network at the cell periphery (7,10,15,16). Indeed, as newborn arteries mature in the immediate postnatal period, the endothelial actin cytoskeleton remodels into a peripheral cortical network and there is a marked increase in endothelium-dependent dilatation (1,3,8,15,16,27). ...
Endothelium of fetal or newborn arteries is atypical, displaying actin stress fibers and reduced NO-mediated dilatation. This study tested the hypothesis that Rho/Rho kinase signaling, which promotes endothelial stress fibers and inhibits endothelial dilatation, contributed to this phenotype. Carotid arteries were isolated from newborn (postnatal day 1, P1), P7 and P21 mice. Endothelial dilatation to acetylcholine (pressure myograph) was minimal at P1, increased at P7 and further increased at P21. Inhibition of Rho (C3 transferase) or Rho kinase (Y27632, fasudil) significantly increased dilatation to acetylcholine in P1 arteries, but had no effect in P7 or P21 arteries. After inhibition of NO synthase (LNAME), Rho kinase inhibition no longer increased acetylcholine responses in P1 arteries. Rho kinase inhibition did not affect dilatation to the NO donor, DEA-NONOate. The endothelial actin cytoskeleton was labelled with phalloidin and visualized by laser scanning microscopy (LSM). In P1 arteries, the endothelium had prominent transcytoplasmic stress fibers, whereas in P7 and P21 arteries, the actin fibers had a significantly reduced intensity and were restricted to cell borders. Phosphorylation of myosin light chains (PO4-MLC), a Rho kinase substrate, was highest in P1 endothelium and significantly reduced in P7 and P21 endothelium (LSM). In P1 arteries, inhibition of Rho (C3 transferase) or Rho kinase (Y27632) significantly reduced the intensity of actin fibers, which were restricted to cell borders. Similarly, in P1 arteries, Rho inhibition significantly reduced endothelial levels of PO4-MLC. These results indicate that the atypical function and morphology of newborn endothelium is mediated by Rho/Rho kinase signaling.
... An inverse relationship between cell differentiation and cytoplasmic actin expression has been observed (28,36). The decrease of the 2.1-kb cytoplasmic actin mRNAs during late spermiogenesis and the predominant expression of the testicular SMGA mRNA in haploid male germ cells show a pattern analogous to those found during the in vivo differentiation of rat aortic smooth muscle, where a decrease in cytoplasmic actin is followed by an increase in smooth muscle a-actin (28). ...
... An inverse relationship between cell differentiation and cytoplasmic actin expression has been observed (28,36). The decrease of the 2.1-kb cytoplasmic actin mRNAs during late spermiogenesis and the predominant expression of the testicular SMGA mRNA in haploid male germ cells show a pattern analogous to those found during the in vivo differentiation of rat aortic smooth muscle, where a decrease in cytoplasmic actin is followed by an increase in smooth muscle a-actin (28). In both cases, cytoplasmic actin decreases as cell differentiation proceeds. ...
... A new IFL substrate based on an embryonal aorta vascular smooth muscle (VSM) cell line has been previously proposed as an easier and more reliable method of identifying actin MF (28,29). The VSM cell line is obtained from the thoracic aorta of rat embryo which expresses characteristic smooth muscle proteins such as cytoplasmic actin of the microfilament network (30,31). The diagnostic accuracy of this assay, however, has not been investigated in a large number of AIH-1 patients and controls. ...
Smooth muscle antibodies (SMA) with anti-microfilament actin (MF-SMA) specificity are regarded as highly specific markers of type 1 autoimmune hepatitis (AIH-1) but their recognition relying on immunofluorescence of vessel, glomeruli, and tubules (SMA-VGT pattern) in rodent kidney-tissue, is restricted by operator-dependent interpretation.A gold standard method for their identification is not available. We assessed and compared the diagnostic accuracy for AIH-1 of an embryonal-aorta vascular-smooth-muscle(VSM) cell line-based assay with those of the rodent-tissue based assay for the detection of MF-SMA pattern in AIH-1 patients and controls. Sera from 138 AIH-1 patients and 295 controls (105 primary biliary cholangitis,40 primary sclerosing cholangitis,50 chronic viral hepatitis,20 alcohol-related liver disease,40 steatotic liver disease,and 40 healthy controls) were assayed for MF-SMA and for SMA-VGT using VSM-based and rodent tissue-based assays, respectively. MF-SMA and SMA-VGT were found in 96(70%) and 87(63%) AIH-1 patients, and 2 controls (p<0.0001).Compared with SMA-VGT, MF-SMA showed similar specificity (99%), higher sensitivity (70% vs 63%,p=ns) and likelihood ratio for a positive test (70 vs 65). Nine (7%) AIH-1 patients were MF-SMA positive despite being SMA-VGT negative. Overall agreement between SMA-VGT and MF-SMA was 87% (kappa coefficient 0.870,[0.789-0.952]). MF-SMA were associated with higher serum γ-globulin [26(12-55) vs 20 g/l(13-34),p<0.005] and immunoglobulin G (IgG) levels [3155(1296-7344) vs 2050 mg/dl(1377-3357), p<0.002]. The easily recognizable IFL MF-SMA pattern on VSM cells strongly correlated with SMA-VGT and has an equally high specificity for AIH-1. Confirmation of these results in other laboratories would support the clinical application of the VSM cell-based assay for reliable detection of AIH-specific SMA.
... Synthetic VSMCs predominate in the embryonic and neonatal periods, and contractile VSMCs (or differentiated cell types) are mainly found in the vessels of adults [7][8][9][10]. Cytoskeletal changes in contractile VSMCs include an increase in the α isoform of actin and the appearance of desmin [24,25]. However, under some pathological conditions, these cells are able to return to a synthetic phenotype (or a dedifferentiated state), which is usually an early event in atherogenesis. ...
Atherosclerosis (AS) is a dynamic and multi-stage process that involves various cells types, such as vascular smooth muscle cells (VSMCs) and molecules such as microRNAs. In this study, we investigated how miR-338-3p works in the process of AS. To determine how miR-338-3p was expressed in AS, an AS rat model was established and primary rat VSMCs were cultured. Real-time polymerase chain reaction was performed to detect miR-338-3p expression. Markers of different VSMC phenotypes were tested by Western blot. Immunofluorescent staining was employed to observe the morphologic changes of VSMCs transfected with miR-338-3p mimics. A dual luciferase reporter assay system was used to verify that desmin was a target of miR-338-3p. To further identify the role of miR-338-3p in the development of AS, VSMC proliferation and migration were evaluated by EdU incorporation assay, MTT assay, and wound healing assay. miR-338-3p expression was upregulated in the aortic tissues of an AS rat model and in primary rat VSMCs from a later passage. The transfection of miR-338-3p mimics in VSMCs promoted the synthetic cell phenotype. Bioinformatics analysis proposed desmin as a candidate target for miR-338-3p and the dual luciferase reporter assay confirmed in vivo that desmin was a direct target of miR-338-3p. The MTT and EdU incorporation assay revealed increased cell viability when miR-338-3p mimics were transfected. The increased expression of PCNA was a consistent observation, although a positive result was not obtained with respect to VSMC mobility. In AS, miR-338-3p expression was elevated. Elevated miR-338-3p inhibited the expression of desmin, thus promoting the contractile-to-synthetic VSMC phenotypic transition. In addition to morphologic changes, miR-338-3p enhanced the proliferative but not mobile ability of VSMCs. In summary, miR-338-3p promotes the development of AS.
... The expression of α-SM actin has been considered as an indicator of the degree of differentiation in smooth muscle cells. Changes in the expression of α-SM actin have been found during the development of the aorta (Kocher et al., 1985), in wound healing (Darbi et al., 1990), in atherosclerotic lesions (Gabbiani et al., 1984;Kocher et al., 1991) and in cultured smooth muscle cells obtained from aortic explants (Chamley-Cambell et al., 1979). During the development of atherosclerotic lesions, both TGF-β1 and PDGF are released from platelets and macrophages (see Introduction). ...
α-Smooth muscle actin is considered a reliable marker for distinguishing between arterial smooth muscle and endothelial cells. Several authors have reported heterogeneity in the expression of this actin isoform in atherosclerotic lesions. Such heterogeneity appears to result from the presence of different smooth muscle cell phenotypes (contractile and synthetic) in these lesions. In the present study, we show that bovine aortic endothelial cells, which are characterised by the presence of Factor VIII-related antigen (FVIII) and by the absence of αsmooth muscle actin (α-SM actin) may be induced to express the latter when exposed to TGF-β1. FVIII was detected by immunofluorescence, α-SM actin was detected by immunofluorescence and immunoblotting. The number of cells expressing α-SM actin increased with time of incubation with TGF-β1, and this increase occurred concomitantly with a decrease in the expression of FVIII. Double immunofluorescence demonstrated the presence of cells that expressed both FVIII and α-SM actin after 5 days of incubation with TGF-β1. With longer incubation times (10-20 days) the loss of FVIII expression was complete and over 90% of the cells expressed α-SM actin.
Ultrastructurally, cells in control cultures showed the typical features of endothelial cells. In the TGF-β1-treated cultures, cells which appeared indistinguishable from contractile and synthetic smooth muscle cells were observed. Withdrawal of TGF-β1 after 10 days incubation resulted in the re-appearance of polygonal cells which were FVIII-positive and α-SM actin-negative. Other cells in the same cultures, however, remained ragged in morphology, FVIII-negative and α-SM actinpositive even after 20 days in control medium. This indicates that the inductive effect of TGF-β1 is partly reversible after 10 days incubation. Such reversibility was no longer apparent after 20 days incubation with TGF-β1.
Our results demonstrate that TGF-β1 induces the differentiation of aortic endothelial cells into a smooth muscle-like phenotype and suggest a novel role for TGF-β1 in atherogenesis.
... We speculate that RUV activates mechanisms of graft healing and aortic vessel wall maturation. It was shown in rats, that there is a constant increase in aortic laminin and actin expression between week 5 and 12 during development [66] . This expression is important for the maturation of the smooth muscle cell phenotype. ...
Vascular grafts with a diameter of less than 6 mm are made from a variety of materials and techniques to provide alternatives to autologous vascular grafts. Decellularized materials have been proposed as a possible approach to create extracellular matrix (ECM) vascular prostheses as they are naturally derived and inherently support various cell functions. However, these desirable graft characteristics may be limited by alterations of the ECM during the decellularization process leading to decreased biomechanical properties and hemocompatibility. In this study, arteries from the human placenta chorion were decellularized using two distinct detergents (Triton X-100 or SDS), which differently affect ECM ultrastructure. To overcome biomechanical strength loss and collagen fiber exposure after decellularization, riboflavin-mediated UV (RUV) crosslinking was used to uniformly crosslink the collagenous ECM of the grafts. Graft characteristics and biocompatibility with and without RUV crosslinking were studied in vitro and in vivo. RUV-crosslinked ECM grafts showed significantly improved mechanical strength and smoothening of the luminal graft surfaces. Cell seeding using human endothelial cells revealed no cytotoxic effects of the RUV treatment. Short-term aortic implants in rats showed cell migration and differentiation of host cells. Functional graft remodeling was evident in all grafts. Thus, RUV crosslinking is a preferable tool to improve graft characteristics of decellularized matrix conduits.
... 51,52 In the embryogenesis, the expression of several gene markers and the production of different proteins such as extracellular matrix protein (ECM) is highly induced. 54,55 In healthy mature organisms, VSMCs are in differentiated state and regulate vascular tone, which is their primary function. 53 Vascular diseases or injury can disturb this differentiated state. ...
Overweight and obesity are chronic diseases, which are increasingly affecting children and adolescents, and if not treated immediately then fat children from today will become patients from tomorrow. The objective of this study was to investigate whether dietary fat restriction normalizes body weight, impaired glucose tolerance and endothelium-dependent contractions induced by high dietary fat intake in young rodents. C57BL/6J mice, 4 weeks of age, were divided into Control group, which was fed with standard rodent chow (12% of fat); High-Fat group, which was fed for 30 weeks with the high-fat (HF) diet (41% fat) and Fat Restriction group, which was fed for 15 weeks with the HF diet (41% of fat), followed by standard chow (12% of fat) for 15 weeks. Body weight was monitored and glucose tolerance test was performed. Vascular responses to acetylcholine were investigated in aortic and carotid artery rings ex vivo in the absence or presence of nitric oxide and prostanoids. Body weight was increased in the High-Fat group and was normalized in the Fat Restriction group to similar levels as in the Control group. Impaired glucose tolerance detected in High- Fat group was normalized in the Fat Restriction group. In the High-Fat group, endotheliumdependent contractions were increased in aorta and carotid arteries, and these contractions were attenuated in the Fat Restriction group to the same level as in the Control group. Moreover, the extent and sensitivity of these contractions varied between aorta and carotid arteries in the presence or absence of nitric oxide. In conclusion, intake of high amounts of fat leads to weight gain, glucose intolerance and enhanced endothelium-dependent contractile responses in aorta and carotid artery of young mice. All these effects were normalized after dietary fat restriction. These findings thus suggest that long term reduction in intake of dietary fat improves the metabolic and vascular function in young mice.
... The development of the human aorta is characterized by nondifferentiated SMC becoming fully differentiated and altering their phenotype several times (14,15,26,35). Signals controlling SMC differentiation remain as yet unknown. ...
A membrane glycoprotein complex was isolated and purified from human smooth muscle by detergent solubilization and affinity chromatography on collagen-Sepharose. The complex was identified as VLA-1 integrin and consisted of two subunits of 195 and 130 kD in SDS-PAGE. Liposomes containing the VLA-1 integrin adhered to surfaces coated with type I, II, III, and IV collagens, Clq subcomponent of the first component of the complement, and laminin. The liposomes specifically adhered to these proteins in a Ca2+, Mg2(+)-dependent manner, but did not bind to gelatin, fibronectin, and thrombospondin substrates. The expression of VLA-1 integrin in different human tissues and cell types, and during aorta smooth muscle development was studied by SDS-PAGE, and subsequent quantitative immunoblotting was performed with antibodies recognizing alpha 1 and beta 1 subunits of the VLA-1 integrin. A high level of VLA-1 integrin expression was an exceptional feature of smooth muscles. Fibroblasts, endothelial cells, keratinocytes, striated muscles, and platelets contained trace amounts of VLA-1 integrin. In the 10-wk-old human fetal aorta, VLA-1 integrin was found only in smooth muscle cells whereas mesenchymal cells, surrounding aortic smooth muscle cells, were VLA-1 integrin negative. By the 24th wk of gestation, the amount of VLA-1 integrin was significantly reduced in the aortic media (4.3-fold for alpha 1 subunit and 2.5-fold for beta 1 subunit) compared with that in the 10-wk-old aortic smooth muscle cells. After birth, the expression of VLA-1 integrin increased and in the 1.5-yr-old child aorta the VLA-1 integrin level was almost the same as in adult aortic media. Smooth muscle cells from intimal thickening of adult aorta express five times less alpha 1 subunit of VLA integrin that smooth muscle cells from adult aortic media. In primary culture of aortic smooth muscle cells, the content of the VLA-1 integrin was dramatically reduced and subcultured cells did not contain VLA-1 integrin at all.
... 10,15 A dynamic equilibrium thus exists between the myofibroblast and SMC phenotype and in pathological conditions the myofibroblast can express desmin and α-SMA indicating a myofibroblast phenotype more closely resembling the SMC. The SMC can also acquire the ultrastructural characteristics of the myofibroblast, 38 supporting the concept that SMCs are progenitors of the myofibroblast. It is suggested that the myofibroblast and SMC are cellular isoforms with a common ancestor cell and that the Fø, together with the SMC, are embryologically derived from the same primitive mesenchymal cell. ...
Intestinal fibrosis is a major complication of the inflammatory bowel diseases (IBD) and although inflammation is necessary for its development, it would appear that it plays a minor role in its progression as anti-inflammatory treatments in IBD do not prevent fibrosis once it has started. The processes that regulate fibrosis would thus appear to be distinct from those regulating inflammation and, therefore, a detailed understanding of these pathways is vital to the development of anti-fibrogenic strategies. There have been several recent reviews exploring what is known, and what remains unknown, about the development of intestinal fibrosis. This review is designed to add to this literature but with a focus on the cellular components that are involved in the development of fibrogenesis and the major molecular mediators that impact on these cells. The aim is to heighten the understanding of the factors involved in intestinal fibrogenesis so that detailed research can be encouraged in order to advance the processes that could lead to effective treatments.
... Portions containing approximately 20 ,ug of protein were loaded into the slots of a 7.5 to 12.5% gradient polyacrylamide-SDS gel before electrophoresis was performed at a constant current of 20 mA in the stacking gel and 40 mA in the running gel. After Coomassie blue staining, gels were scanned with a high-resolution laser beam densitometer connected to a microcomputer (13). The relative quantities of the major outer membrane proteins were determined fromn at least three different preparations. ...
... For control staining procedures, the primary antibody was omitted. Furthermore, adjacent myocardial and central nervous system Kocher et al. (1984) DV A rat Kocher et al. (1985) DV A rat 1 D, desmin; V, vimentin; C, cytokeratin; H, human; A, animal. tissue served as internal controls of the immunohistochemical stainings. ...
Background
The cytoskeleton of cells in blood vessel walls contains desmin, vimentin, and cytokeratins. The distribution of these proteins in human vessels is not fully known. We have mapped the distribution of intermediate filament proteins in human arterial walls.Methods
Monoclonal antibodies targeted at the intermediate filament proteins desmin, vimentin, and cytokeratins were used, and the distribution of these proteins was studied by immunohistochemistry.ResultsIn the muscular arteries, most smooth muscle cells in the media expressed both desmin and vimentin; in the elastic arteries, the proportion of desmin-labelled cells was lower and preferentially located to the periphery of the media. In general, the desmin immunoreactivity within the intima was weak, but some smooth muscle cells and smooth muscle cells in the musculoelastic layer showed strong immunoreactivity. The vasa vasorum exhibited a heterogeneous desmin-labelling pattern. The vimentin antibodies labelled the endothelium and showed a heterogeneous staining pattern in the other layers of the arterial wall. Cytokeratin was detected in occasional cells in the media of muscular arteries, in many adluminal cells and cell clusters in the coronary intima, and in smooth muscle cells in the media of the elastic arteries.Conclusions
Vimentin is widely distributed in vascular smooth muscle cells, whereas the distribution of desmin and cytokeratin varies. Each artery studied had an intermediate filament pattern typical for the anatomical location. There were no interindividual variations in the distribution of intermediate filament proteins. Anat. Rec. 247:439–448, 1997. © 1997 Wiley-Liss, Inc.
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.
L'augmentation de la fréquence des pathologies vasculaires va créer ces prochaines années, des besoins importants en substituts vasculaires de petits calibres. L'idéal, à ce jour, reste l'utilisation de vaisseaux autologues réduisant les risques infectieux et immunologiques mais d'usage limité ce qui est à l'origine de l'ingénierie cellulaire. L'identification des progéniteurs vasculaires est d'un intérêt majeur dans ce domaine. Ces cellules d'origine autologues sont douées d'une grande capacité de prolifération et d'un potentiel de différenciation en cellules vasculaires (cellules endothéliales (CE) et cellules musculaires lisses (CML)) mais la difficulté réside dans le choix du recouvrement de la surface du support de culture qui favorise leur adhésion et prolifération Dans ce travail, nous avons choisi les films multicouches de polyélectrolytes et mesuré leur impact sur le comportement de ces cellules progénitrices en fonction de différentes conditions de culture (normoxie ou hypoxie). Nous avons montré dans un premier temps, que ces films multicouches de polyélectrolytes permettaient d?accélérer la différenciation de ces cellules en CE matures. Nous avons également montré que ces mêmes cellules cultivées en hypoxie étaient capables de se différencier en cellules contractiles stables dans le temps, présentant un phénotype comparable à celui des CML matures. L'association de ces résultats additionnés aux avantages apportés par des feuillets détachables est à la base de la construction d'un substitut vasculaire autologue de petit calibre, composé de CE et de CML issues d'un même pool de cellules, mais cultivées dans des conditions différentes.
Normal muscular and elastic arteries consist of three morphologically distinct layers [20,325] which will be considered in more detail in Chap.4. The intima consists of a narrow region bounded on the luminal side by a single continuous layer of endothelial cells and peripherally by a fenestrated sheet of elastic fibres, the internal elastic lamina. Between these boundaries are smooth-muscle cells and various components of extracellular connective-tissue matrix. With increasing age in man, intimal smooth-muscle cells and extracellular matrix components accumulate slowly and generally at a uniform rate, except in certain areas where nodular accumulations called “cushions” develop (see Chap 3). Why so few smooth-muscle cells are initially present in the intima, and why the number of these cells normally increases with age, are important questions that remain to be answered.
We investigated the mechanism of heparin-derived oligosaccharide on the proliferation of vascular smooth muscle cell (VSMC) induced by vascular endothelial growth factor (VEGF). Expression levels of VEGFR 1 and VEGFR 2 were examined by RT-PCR, and the corresponding protein expression levels were detected by Western blotting and immunocytochemistry. Western blotting was taken to identify the expression levels of mechanism proteins. The binding of VEGF and VEGFR 2 was measured by co-IP. Besides, HS competition assay was to detect the ability of HDO to compete with Heparin for VEGF165. HDO showed an inhibitory effect on the expression of VEGFR1/2 proteins and PKC, MAPK, PI3K/Akt pathways. In addition, HDO affected the binding of VEGF-VEGFR, which may be one of the most important mechanisms of HDO suppress the cell proliferation induced by growth factors. Thus HDO showed the ability as a VEGF antagonist.
The aims of the present study were to determine the effects of heparin-derived oligosaccharides (HDOs) on vascular intimal hyperplasia (IH) in balloon-injured carotid artery and to elucidate the underlying mechanisms of action. An animal model was established by rubbing the endothelia within the common carotid artery (CCA) in male rabbits. The rabbits were fed a high-cholesterol diet. Arterial IH was determined by histopathological changes to the CCA. Serum lipids were detected using an automated biochemical analysis. Expressions of mRNAs for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), scavenger receptor class B type I (SR-BI), and ATP-binding cassette transporter A1 (ABCA-1) were analyzed using reverse transcription polymerase chain reaction assays. Expressions of VEGF, VCAM-1, MCP-1, SR-BI and ABCA-1 proteins were analyzed by Western blotting. Enzyme-linked immunosorbent assays were used to quantify expression levels of VEGF and bFGF. Our results showed that administration of HDO significantly inhibited CCA histopathology and restenosis induced by balloon injury. The treatment with HDOs significantly decreased the mRNA and protein expression levels of VEGF, bFGF, VCAM-1, MCP-1, and SR-BI in the arterial wall; however, ABCA-1 expression level was elevated. HDO treatment led to a reduction in serum lipids (total cholesterol, triglycerides, high-density and low-density lipoproteins). Our results from the rabbit model indicated that HDOs could ameliorate IH and underlying mechanism might involve VEGF, bFGF, VCAM-1, MCP-1, SR-BI, and ABCA-1.
The production of human matrix-degrading enzymes by cultured smooth muscle cells (SMC) from the human aortic intima was investigated by using antibodies against matrix metalloproteinases that had been purified from human rheumatoid synovial fibroblasts. Like the DNA synthesis of SMC, the platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) stimulated SMC to produce matrix metalloproteinase 1 (MMP-1) and 3 (MMP-3). The increased production of MMP-1 and 3 by PDGF, however, was inhibited by the addition of dexamethason, vitamin A, and estradiol to the cultures. These results suggested that the ability of SMC to produce MMP-1 and 3 was related to its proliferation and/or migration in vivo.
On the other hand, linoleic acid hydroperoxide inhibited the DNA synthesis of SMC, although it stimulated the cells to produce MMP-1 and 3, suggesting that the effect of lipid peroxide on the production of MMPs might disrupt the physiological turnover of extracellular matrix macromolecules.
The large amounts of MMP-2 (gelatinase/so called type IV collagenase) were constitutively synthesized in the cultured SMC and the effect of PDGF or EGF on by SMC production of MMP-2 was negligible.
Adseverin of gelsolin family was distributed in neuronal cells, endocrinal cells and blood cells with exocytic function, but not in mormal adult smooth muscle cells. In primary culture of smooth muscle from bovine carotid artery, we found that this protein had gradually been expressed, arriving at maximum in confluent state by immunoblotting method.And then, we examined the expression of this protein in cultured cells time-dependently by immunofluorescent method as comparing with other structural proteins.
On 1st day of cultured cell by enzymatic preparation without serum, adseverin was not expressed in all the cells with deformed globular feature, but smooth muscle type of alpha-actin was clearly expressed. On 4th day after addition of serum, adseverin was expressed at the surface membrane of all the globular cells. In this state bipolar cells appeared under the globular cells in certain place. These cells had neither adseverin nor alpha-actin. Once bipolar cells appeared, their growth rate was very fast their numbers were rapidly increasing. Adseverin and alpha-actin were still not expressed in these cells, but stress fibers were observed by rhodamine phalloidin. When cells were going on confluent state, accompanied by post-confluent state with hills and valleys structure, globular cells were gathered, and formed tight round structure on the top of the hill.
On the other hand, in tissue of carotid artery adseverin was not detected in smooth muscle at the media, but at the hypertrophic area of intima adseverin was observed in small numbers of the cell. From these results we would like to propose the idea that there is a kind of stem cell of undifferentiated smooth muscle in the hypertrophic area of intima, and that these cells with globular form could be divided, but their cell cycle was very slow. Soon after different type of cell, perhaps undifferentiated cell with bipolar feature was separated and its growing rate was very fast. We discovered the stem cell of undifferentiated smooth muscle in bovine carotid artery primary cell culture and adseverin is an excellent marker of the cell, which means that this stem cell produce actively some growth factors using adseverin for exocytic function.
Under physiological conditions, the arterial endothelium exerts a powerful protective influence to maintain vascular homeostasis. However, during the development of vascular disease, these protective activities are lost and dysfunctional endothelial cells actually promote disease pathogenesis. Numerous investigations have analyzed the characteristics of dysfunctional endothelium with a view to understanding the processes responsible for the dysfunction and to determining their role in vascular pathology. The present review adopts an alternate approach: reviewing the mechanisms that contribute to the initial formation of a healthy protective endothelium and on how those mechanisms may be disrupted, precipitating the appearance of dysfunctional endothelial cells and the progression of vascular disease. This approach, which highlights the role of endothelial adherens junctions and VE-cadherin in endothelial maturation and endothelial dysfunction, provides new insight into the remarkable biology of this important cell layer and its role in vascular protection and vascular disease.
The TGF-β superfamily consists of a large number of structurally related cytokines, including activin/inhibin, the bone morphogenetic proteins (BMPs), the growth and differentiation factors (GDFs), the TGF-βs and a number of proteins involved in developmental patterning. Much of the research into the biology of the TGF-β superfamily has focused on TGF-β1 (the first to be discovered), and it is this isoform about which most is known.
Changes in smooth muscle α-actin expression in an organ culture system was examined by bidimensional gel electrophoresis (2-D PAGE) and isoelectric focussing (IEF).
It has been shown that in normal arteries smooth muscle cells predominantly express smooth muscle α-actin whereas in cultured smooth muscle cells aswitch in actin-isoform pattern to a predominance of cytoplasmatic ß-actin has been observed. Therefore we examined whether this phenomenon also occurs in the media of cultured aorta segments (organ cultures). These segments can be maintained in culture without loss of endothelium and contractility. After a period of three, seven and eleven days in culture no reduction of smooth muscle α-actin content was observed compared with normal rabbit thoracic aorta despite maintenance in 30% calf serum. Even denudation of the rabbit thoracic aorta prior to cultivation did not cause a decrease in smooth muscle α-actin content. We conclude that three dimensional structure and extracellular matrix components of organ culture segments play a major role in maintenance of smooth muscle α-actin expression.
The basic process of blood vessel development is not well understood. It is known that vessel formation is initiated by the aggregation of endothelial precursors (angioblasts) as well as by the invasion of endothelial cells into existing tissue.1 Subsequently, there is recruitment of cells from the surrounding mesenchyme that give rise to smooth muscle cells (SMCs).2,3 Additionally, in medium and large vessels, there is first continued proliferation of SMCs, organization of these cells into distinct layers separated by elastic laminae, and eventual cessation of growth.4,3,5,6 This last phase of blood vessel development, which we have termed “developmental maturation,” defines the transition of SMCs from a proliferative to a contractile phenotype. Research interest in this phase of vascular development arise from the morphological and biochemical similarities that have been noted between fetal SMCs and SMCs in atherosclerotic plaques.7,8,9,10,11,12 These similarities have led to the suggestion that SMCs in atherosclerosis and hypertension may recapitulate certain aspects of earlier developmental events.9,13 To permit a critical analysis of this possibility, it is crucial that we obtain a better understanding of the developmental events associated with the transition of SMCs from a proliferative to a quiescent phenotype. As a first step toward this goal, we have compared these two populations of SMCs and have identified genes that are specifically expressed in the fetal SMC population and not in the adult SMCs.
Despite intensive clinical, experimental and epidemiological studies, the mechanisms leading to the formation of the lesions of atherosclerosis remain mysterious. In the last few years, however, significant progress has been made in understanding the roles played by arterial wall components, blood-borne cells and plasma constituents in the development of the atheromatous plaque [for reviews see 4,43,49,63,90,92,93,130]. This process appears more and more to depend on interactions among various causative factors, thus making the search for a prime cause particularly difficult and possibly pointless. In this chapter, we shall limit our discussion to the role of cellular elements, particularly those normally present in the vessel wall, in the development of the atheromatous plaque.
Numerous are the reports on the preparation techniques of human saphenous vein grafts in order to maximize the rate of graft patency after implantation. The aim of all methods is to preserve the morphological as well as the functional integrity of the vein wall. One of the most important layers in the vein wall is the endothelium as it forms the barrier between the intravascular and extravascular compartment. The preservation of the endothelium is the major goal of all preparation techniques.1–4 The endothelial cells are selective in their permeability, form a nonthrombogenic surface, have several receptors for low density lipoproteins, produce growth factors and are able to produce and secrete several vasoactive substances.5 All histopathological, surgical and pharmacological trials study the alteration of the metabolism and the response of the endothelium to injury during preparation.2,4,6–9 Despite the wide-spread use of the saphenous veins to bypass narrowed coronary arteries, the morphological changes in human vein grafts occurring after harvesting and in the first days after operation have only briefly been reported in vein graft literature.10–14In most of these studies it is assumed that no significant lesions can be found in the grafts which have been in place for less than 2 weeks.15–17
Cytoskeletal and cytocontractile elements are involved in many cell physiological processes such as maintenance of cell shape, locomotion, mitosis, and secretion (for review see Alberts et. al., 1989). Moreover, the study of these structures may contribute to the understanding of cellular alteration during pathological situations (for review see Rungger-Brändle and Gabbiani, 1983). Different types of cytoskeletal changes may cause pathological situations. In some instances, a disease is due to the presence of an abnormal cytoskeletal protein. A well-documented example of this is the immotile cilia syndrome, characterized by an impaired mucociliary transport leading to chronic infections of airways (Camner et. al., 1975; Eliasson et. al., 1977); here, the basic defect is due to abnormal or missing dynein arms (Afzelius, 1985; Neustein et. al., 1980; Pedersen and Mygind, 1976). In other situations the primary defect affects a noncytoskeletal cellular component but leads to typical changes of the cytoarchitecture, as is the case for sickle-cell anemia where the primary defect is a mutant β-globin molecule leading to an altered organization of the cytoskeleton during the sickling process (Lux, 1979). In most instances, however, changes in the organization of cytoskeletal elements associated with pathological conditions reflect the degree of cellular adaptation to pathological stimuli (Rungger-Brändle and Gabbiani, 1983). Thus, cytoskeletal organization can be profoundly modified by pathological processes affecting cell activities such as migration or proliferation. Moreover, certain important pathological processes, such as tumor invasion, may at least in part depend on altered cytoskeletal organization (for review see Boschek, 1982).
This chapter reviews current concepts related to the formation of vascular beds in the normal lung at different stages of growth and maturation, comments briefly on aberrant growth that results in a still adequately functioning lung, and summarizes the lung vasculature in aging. It also highlights the presence of signaling systems that, triggered by the ambient oxygen tension, change vascular density in the adult lung. The vascular units of the lung form in close coordination with airways and alveoli. Lung vascular growth is achieved by expansion of existing structures and by change in existing templates. As vascular networks increase in size and three-dimensional complexity, growth is accompanied by regression of unneeded units until these are appropriate to the stage of lung development. In this way, vascular systems formed in utero, in the postnatal period, and in early childhood, develop, enlarge and are remodeled. In the human embryo, the formation of vascular networks starts with the onset of organogenesis in the fourth week of gestation. As the lung anlage develops from the foregut, it is vascularized by ingrowth of a vascular plexus derived from the heart and the main pulmonary artery develops from migrating angioblasts. At 36 weeks to term of human gestation, the lung responds by a burst of vascular growth as existing units continue to expand in diameter and length, and many new intra-acinar units are added with formation of the gas-exchange surface. In the first months of postnatal life, the diameter of proximal intra-acinar vessels increases more than distal ones reflecting the burst of small vessels developing at the lung periphery.
The process of wound repair is of vital importance for animals as well as for plants (Shigo, 1985), since a wound perturbs body homeostasis and may result in infection by microorganisms. A wound may occur without or with tissue loss (Robbins et al, 1984). In both cases, but more clearly in the second case, wound healing consists schematically of acute inflammation followed by formation of granulation tissue, a transitional tissue able to retract the wound space, and finally scar formation.
Vascular growth in the developing lung proceeds in a series of intricate steps that include the expansion of existing structures and changes to existing templates. As vascular networks increase in size, and three-dimensional complexity, their growth is accompanied by the regression of unneeded units until those formed are appropriate to age and stage of lung development. In this way, the vascular systems formed in utero, in the postnatal period, and in early childhood develop, enlarge and remodel; and in late childhood, and in the young adult, continue to enlarge and remodel until thoracic growth is complete. This chapter focuses on current concepts of morphogenesis and signaling pathways within the lung’s forming vascular networks, describes studies in the human lung at different stages of growth and maturation, comments briefly on the little known about the lung vasculature in aging, and summarizes examples of aberrant growth that result in a still adequately functioning lung.
The primary function of smooth muscle is to produce the contraction phenomena in the circulatory, respiratory, gastrointestinal, and urogenital systems. However, in the adult organism the smooth muscle cell retains significant proliferative, synthetic, and secretory functions compared to skeletal and cardiac myocytes. The variety of physiological roles played by smooth muscle cells in the body explains the heterogeneity of their functional and anatomical features. Although smooth muscle heterogeneity has been established for a long time, the definition criteria have been refined only during the past few decades. As of today, the expression of smooth muscle cell-specific proteins, in particular the relative expression of α- and γ-smooth muscle actins, is the best criterion to appraise heterogeneity of enteric and vascular smooth muscles, as well as of smooth muscle-like cells such as myofibroblasts, myoepithelial and myoid cells. Investigating the heterogeneity and plasticity characteristics of smooth muscle cells is of major importance for understanding the mechanisms involved in their normal and pathological adaptations.
Arteries act as conduits for blood, but this is not their only function; much of their specialized structure is dependent on the essential physiological role they have to play in the circulation. They develop under two sets of influences: genetic factors mainly control the morphological pattern of the circulation, and haemodynamic factors control the form of the vessel wall. The haemodynamic factors, permit wall modifications that preserve certain mechanical properties [1–5]. The necessity to preserve these properties, an essential part of the wall function, determines much of the vascular response to hypertension and probably the interindividual variation in vessel wall thickness, in direct relationship to individual blood pressure, as mentioned in Chapter 1.
The late parental phase of mammalian blood-vessel development is a complex and poorly understood process that involves dynamic changes in smooth-muscle structure and function. This chapter describes the process in terms of cellular-maturation events to emphasize two components of this transition. First, that it is a process that occurs over a relatively long period (1–3 weeks in small mammals). Second, that there is not a “synthetic” versus “contractile” phenotype, but rather, there is a gradual increase in the amount of morphologically recognizable structures associated with a contractile smooth muscle cell. During the earlier phases of prenatal development, highly biosynthetic smooth muscle cells are observed to also accumulate myofilaments and are elastogenic. A reasonable interpretation of this observation is that these cells are now dedicated toward the synthesis of proteins necessary for the contractile machinery.
Platelet-derived growth factor (PDGF) is a dimer composed of two homologous polypeptide chains A and B (Johnsson et al. 1982; Heldin et al. 1985). These peptides are encoded by separate genes localized to different chromosomes (Betsholtz et al. 1986; Swan et al. 1982) and are independently regulated (Betsholtz et al. 1986). It is not clear whether AA, AB, or BB dimers differ in their biological activity. Clearly, some cell types show preferential binding of AA versus BB or AB (Kazlauskas et al. 1988) and this has been attributed to differences in PDGF receptor subtypes synthesized by these cells.
As a general rule, smooth muscle cells (SMC) are able to switch from a contractile phenotype to a less mature synthetic phenotype. This switch is accompanied by a loss of differentiation with decreased expression of contractile markers, increased proliferation as well as the synthesis and the release of several signaling molecules such as pro-inflammatory cytokines, chemotaxis-associated molecules and growth factors. This SMC phenotypic plasticity has extensively been investigated in vascular diseases but interest is also emerging in the field of gastroenterology. It has in fact been postulated that altered micro-environmental conditions, including the composition of microbiota, could trigger the remodeling of the enteric SMC, with phenotype changes and consequent alterations of contraction and impairment of gut motility. Several molecular actors participate in this phenotype remodeling. These include extracellular molecules such as cytokines and extracellular matrix proteins, as well as intracellular proteins, e.g. transcription factors. Epigenetic control mechanisms and miRNA have also been suggested to participate. In this review key roles and actors of smooth muscle phenotypic switch, mainly in GI tissue, are described and discussed in the light of literature data available so far. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Publisher Summary
This chapter illustrates the expression patterns of several cytoskeletal, contractile, extracellular matrix proteins, and integrins in smooth muscle cells of developing, adult, and atherosclerotic human aorta. This chapter intends to describe the phenotypic properties of smooth muscle cells during development and in disease states; to characterize the diversity of human vascular smooth muscle cells; and to analyze possible strategies for the regulation of individual gene expression used during smooth muscle development and phenotypic transitions of smooth muscle cells in adult, and the role of genetic (intrinsic) and environmental (extrinsic) factors in smooth-muscle-cell phenotypic expression. Whereas intrinsic control plays a major role in the early events of smooth-muscle-cell differentiation, extrinsic factors probably serve as signals for morphogenetic events and determine the phenotypic transitions of mature smooth muscle cells. This chapter focuses on analyzing the coordinate changes in the expression of extracellular matrix components, laminin, fibronectin, extracellular matrix receptors, integrins, and cytoskeletal differentiation markers of smooth muscle cells.
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.
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)
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.
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.
Summary Fat storing cells (FSCs) in the liver represent the main site of vitamin A deposition in the body. These cells are considered
to play an important role during scar formation and fibrogenesis in the liver. The putative descent of FSCs from the fibroblastic
or from the myofibroblastic system have not been determined yet by morphological or immunohistochemical studies. To further
define the origin of these liver cells, we analysed the pattern of expression of three structural proteins: vimentin, desmin
and the α-smooth muscle (SM)-actin isoform in FSCs of the rat liver, in smooth muscle cells (SMCs) from the aorta and in rat
skin fibroblasts. FSCs were studied by immunohistochemical methods immediately after isolation, at days 3 and 7 after plating.
FSC-geneexpression was also analysed by Northern blot analysis of total RNA extracted from cells in culture at days 3 and
7 after isolation. Arterial SMCs and skin fibroblasts were studied in a similar way. For comparison, isolated rat hepatocytes
and Küpffer cells (Kc) were studied. Of freshly isolated FSCs, 100% were vimentin-positive, 50% were desmin-positive, but
all were α-SM-actin negative. Three days after isolation, FSCs were clearly positive for vimentin and desmin and weakly α-SM-actinpositive,
as demonstrated by indirect immunofluorescence as well as by the immunoperoxidase technique. Desmin, α-SM-actin and vimentin
staining was further increased at day 7 after isolation, and α-actin specific transcripts in FSC-RNA were clearly detectable
at day 7 after isolation. Passaged arterial SMCs were vimentin-and α-SM-actin-positive, but desmin-negative and fibroblasts
were only vimentin-positive. α-SM-actin isoform gene expression was strongly increased in the rat liver after acute or chronic
damage induced by treatment with carbon tetrachloride. Positive staining was detected in cells which were also desmin-positive.
As FSCs are vimentin-, desmin-and α-SM-actin-positive, we suggest that they are smooth muscle-related myo-fibroblasts with
special functions.
Experiments investigated maturation of endothelial function in the postnatal period (P). Carotid arteries isolated from newborn (P day 1, P1) to P21 mice were assessed in myographs at transmural pressure (PTM) of 20mmHg (P1 blood pressure, BP). Acetylcholine was ineffective in P1 but powerfully dilated P7 arteries, whereas NO-donor DEA-NONOate caused similar dilation at P1 and P7. Dilation to acetylcholine at P7 was abolished by inhibition of NO synthase (NOS) (L-NAME) or of phosphoinositide-3-kinase (PI3K) (wortmannin, LY294002). Endothelial NOS (eNOS) expression decreased in P7 compared to P1, although acetylcholine increased PO4-eNOS-Ser(1177) in P7 but not in P1 arteries. Endothelial maturation may therefore reflect increased signaling through PI3K, Akt and eNOS. Systemic BP increases dramatically in the early postnatal period. After exposing P1 arteries to transient increased PTM (50mmHg, 60mins), acetylcholine caused powerful dilation and increased PO4-eNOS-Ser(1177). Pressure-induced rescue of acetylcholine dilation was abolished by PI3K or NOS inhibition. Transient increased PTM did not affect dilation at P7, or dilation to NO-donor in P1 arteries. Width of endothelial Adherens Junctions (VE-cadherin immunofluorescence) increased significantly from P1 to P7, and in P1 arteries exposed to transient increased PTM. A function-blocking antibody to VE-cadherin reduced the pressure-induced rescue of acetylcholine dilation at P1, and the dilation to acetylcholine in P7 arteries. Therefore, maturation of newborn endothelium dilator function may be induced by increasing BP in the postnatal period. Furthermore, this may be mediated by VE-Cadherin signaling at Adherens Junctions. Interruption of this maturation pathway may contribute to developmental and adult vascular diseases.
Purpose:
In this study, the effect of heparin-derived oligosaccharide (HDO) on vascular endothelial growth factor (VEGF) induced vascular smooth muscle cell (VSMC) proliferation and the signal transduction mechanisms involved were investigated.
Methods:
MTT assays were used to measure VSMC proliferation, flow cytometry to analyze cell cycle distribution, RT-PCR for detection of gene transcript levels, and cell-based ELISA, Western blotting and immunocytochemical methods to detect the expression of PKC-α, ERK 1/2, p-ERK 1/2, Akt, p-Akt, p-PDK1 and p-GSK-3β.
Results:
HDO at concentrations of 0.01, 0.1 and 1 μmol·L(-1) dose-dependently inhibited VEGF-induced VSMC proliferation with inhibition indices of 6.8 %, 13.1 % and 28.9 %, respectively. Similar concentrations of HDO dose-dependently decreased the percentage of VEGF-induced cells in S phase to 3.6 %, 3.4 %, and 5.4 %, while increasing that of cells arrested in the G0/G1 phase to 80 %, 82 % and 83.6 %. HDO at 0.01, 0.1 or 1 μmol·L(-1) inhibited VEGF-induced PKC-α mRNA expression, with inhibition indices of 9.2 %, 16.1 % and 54.0 %. HDO at 0.1 or 1 μmol·L(-1) inhibited VEGF-induced proto-oncogene mRNA expression, with inhibition indices of 5.2 % and 6.6 % for c-jun, 8.8 % and 11.6 % for c-myc, and 6.5 % and 11.9 % for c-fos, respectively. Additionally, treatment with 0.01, 0.1 or 1 μmol·L(-1) HDO, inhibited VEGF-induced expression of some proliferation related proteins with inhibition indices of 33.2 %, 56.3 % and 77.0 % for PKC-α, 33.7 %, 38.7 % and 53.2 % for p-Akt, 3.5 %, 24.2 % and 49.3 % for p-ERK 1/2, 39.2 %, 71.8 % and 80.7 % for p-PDK 1 and 41.4 %, 89.4 % and 92.4 % for p-GSK-3β, respectively. The results showed that HDO inhibited PKC-α, c-jun, c-fos and c-myc mRNA transcription, and also down-regulated phosphorylation levels of ERK 1/2 and Akt.
Conclusion:
Our study demonstrates that HDO inhibits transcription of proliferation-related proto-oncogenes and arrests G1/S transition through inhibition of the PKC, MAPK and Akt/PI3K pathways in association with inhibition of VSMC proliferation. This altered molecular signature may explain one mechanism of HDO-mediated inhibition of VSMC proliferation.
In order to understand some of the cellular mechanisms of interaction in secondary pulmonary vaso-occlusive disease, we studied 21 lung biopsies from patients with different types of congenital cardiac defects. Their ages ranged from four to 248 months (mean 71.5 months; median 41 months). Changes in the cytoskeleton and extracellular matrix were assessed in the arterial wall. Immunostaining was applied to formalin-fixed, paraffin- embedded tissue, using antibodies to muscle-specific actin, vimentin and fibronectin in supra-optimal dilution. The staining for muscle-specific actin in the medial layer revealed a heterogenous pattern, with areas exhibiting low or absent labelling, reflecting a process of dedifferentiation of the smooth muscle cells in those segments. Within intimal proliferative lesions, the expression of muscle-specific actin was variable, being weak in some lesions and strong in those showing concentrically arranged intimal smooth muscle cells, suggesting a reversion of the migrated cells to the contractile phenotype. The endothelial cells of arteries from cases presenting severe qualitative lesions exhibited strong expression of vimentin, reflecting their heightened regenerative activity and/or their necessity to maintain their shape. The expression of fibronectin was greater in the predominantly cellular lesions of the intima when compared to the fibrotic lesions, indicating the role of that matrix glycoprotein in cellular migration and in replicative processes.
Adhesive interactions are recognized requirements for cellular proliferation, migration and differentiation during normal morphogenesis as well as disease. By differential cloning, osteopontin was identified as an adhesive protein upregulated during vascular remodeling and neointima formation in both rat models and human vascular diseases including atherosclerosis and restenosis. In functional studies, purified osteopontin promoted adhesion, focal contact formation, and migration of vascular smooth muscle and endothelial cells. Utilizing neutralizing antibodies, three integrin-type receptors, alpha v beta 3, alpha v beta 1, and alpha v beta 5 were found to support cellular adhesion to osteopontin. In contrast, only cells containing the alpha v beta 3 integrin could migrate towards an osteopontin gradient, demonstrating for the first time that different functions of osteopontin are mediated via distinct receptors. These results suggest a model whereby osteopontin, via its integrin-type receptors, contributes to vascular remodeling during development and disease by facilitating smooth muscle migration and simultaneously promoting endothelial coverage of the affected area.
Excessive Airway NarrowingPathological Changes in Asthma Affecting Airway Smooth MuscleMechanics of Human Airway Smooth MuscleExperimentally Increased Shortening of Airway Smooth MuscleProliferation of Airway Smooth Muscle in Asthma-Fact or Artefact?Human Asthmatic Smooth-Muscle ResponsesConclusion
References
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.
A procedure for dissociating the rabbit aorta into single, functional smooth muscle cells is described. After removal of adventitia and intima, slices of media were incubated with purified collagenase, elastase, and soybean trypsin inhibitor in a Krebs-Ringer buffer modified with Hepes, amino acids, and a [Ca2+] of 0.2 mM. After enzymatic digestion and mechanical shear, the yield of dispersed cells was approximately 25% based on DNA recovered. Greater than 95% of the cells excluded trypan blue and approximately 80-90% adhered to tissue culture dishes. By phase contrast microscopy, most of the cells were elongate and approximately 10 micron X 30 micron in size. The remainder were either spherical or highly crenated and contracted. Electron microscopy of the cells showed that immediately after dissociation greater than 95% could be identified as smooth muscle, though most had undergone some degree of structural change compared to cells in situ. Depending on the preparation, from 5 to 50% of these cells contracted in response to agonists. Cells shortened by 10-15% and developed numerous evaginations when stimulated by angiotensin II norepinephrine, or carbamylcholine. Cells relaxed after washout of agonists and could subsequently be restimulated. Specific inhibitors of each of the agonists blocked the contractile response. Dispersed cells cultured for 1-5 days contracted in even higher numbers than the freshly prepared cells, suggesting restoration of hormone binding and/or contractile function in culture. This preparation provides a system in which the physiology of individual vascular smooth muscle cells may be studied.
Desmin is a 50,000-mol wt protein that is enriched along with 100-A filaments in chicken gizzard that has been extracted with 1 M KI. Although 1 M KI removes most of the actin from gizzard, a small fraction of this protein remains persistently insoluble, along with desmin. The solubility properties of this actin are the same as for desmin: they are both insoluble in high salt concentrations, but are solubilized at low pH or by agents that dissociate hydrophobic bonds. Desmin may be purified by repeated cycles of solubilization by 1 M acetic acid and subsequent precipitation by neutralization to pH 4. During this process, a constant nonstoichiometric ratio of actin to desmin is attained. Gel filtration on Ultrogel AcA34 in the presence of 0.5% Sarkosyl NL-97 reveals nonmonomeric fractions of actin and desmin that comigrate through the column. Gel filtration on Bio-Gel P300 in the presence of 1 M acetic acid reveals that the majority of desmin is monomeric under these conditions. A small fraction of desmin and all of the actin elute with the excluded volume. When the acetic acid is removed from actin-desmin solutions by dialysis, a gel forms that is composed of filaments with diameters of 120-140 A. These filaments react uniformly with both anti-actin and anti-desmin antiserum. These results suggest that desmin is the major subunit of the muscle 100-A filaments and that it may form nonstoichiometric complexes with actin.
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.
The distribution of actin stress fibers in normal and regenerating (after endothelial denudation by means of a balloon catheter) rabbit aortic endothelial cells has been studied by means of immunofluorescence with human actin autoantibodies on en face endothelial cell preparations. Our results show that: (i) under normal conditions actin is accumulated as a network at the periphery of endothelial cells. Stress fibers are present only in endothelial cells located immediately below intercostal artery branches; (ii) stress fibers develop in endothelial cells early during regeneration and persist after the end of endothelial mitotic and motile activities; and (iii) the orientation of stress fibers within the cytoplasm follows the direction of blood flow, with the exception of stress fibers situated in cells at the edge of the wound, when endothelial cell progression toward the denuded area as well as mitotic activity have ceased. We conclude that stress fibers are an organelle present in endothelial cells in vivo and that they reorganize during endothelial cell adaptation to unfavorable or pathological situations.
The organization of actin and myosin in vascular endothelial cells in situ was studied by immunofluorescence microscopy. Examination of perfusion-fixed, whole mounts of normal mouse and rat descending thoracic aorta revealed the presence of axially oriented stress fibers containing both actin and myosin within the endothelial cells. In both species, the proportion of cells containing stress fibers varied from region to region within the same vessel. Some endothelial cells in mouse mesenteric vein and in rat inferior vena cava also contained stress fibers. Quantitative studies of the proportion of endothelial cells containing stress fibers in the descending thoracic aorta of age-matched normotensive and spontaneously hypertensive rats revealed significant differences. When animals of the same sex of the two strains were compared, the proportion was approximately two times greater in the spontaneously hypertensive rats. The proportion of endothelial cells containing stress fibers was about two times greater in males than in females of both strains. These observations suggest that multiple factors, including anatomical, sex, and hemodynamic differences, influence the organization of the endothelial cell cytoskeleton in situ.
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.
Smooth muscle cells of the digestive, respiratory, and urogenital tracts contain desmin as their major, if not exclusive, intermediate-size filament constituent and also show a predominance of gamma-type smooth muscle actin. We have now examined smooth muscle tissue of different blood vessels (e.g., aorta, small arteries, arterioles, venules, and vena cava) from various mammals (man, cow, pig, rabbit, rat) by one- and two-dimensional gel electrophoresis of cell proteins and by immunofluorescence microscopy using antibodies to different intermediate-sized filament proteins. Intermediate-sized filaments of vascular smooth muscle cells contain abundant amounts of vimentin and little, if any, desmin. On gel electrophoresis, vascular smooth muscle vimentin appears as two isoelectric variants of apparent pI values of 5.30 and 5.29, shows the characteristic series of proteolytic fragments, and is one of the major cell proteins. Thus vimentin has been demonstrated in a smooth muscle cell present in the body. Vascular smooth muscle cells are also distinguished by the predominance of a smooth muscle-specific alpha-type actin, whereas gamma-type smooth muscle actin is present only as a minor component. It is proposed that the intermediate filament and actin composition of vascular smooth muscle cells reflects a differentiation pathway separate from that of other smooth muscle cells and may be related to special functions and pathological disorders of blood vessels.
We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.
The presence of intracellular keratin was examined in 230 human neoplasms using indirect immunofluorescence on fresh frozen, acetone-fixed sections. The use of antikeratin antibodies raised in rabbits against human callus and purified by affinity chromatography proved to be a rapid, sensitive, and reliable method of demonstrating keratin. Epithelial tissues and epithelial-derived neoplasms were found to contain keratin, whereas tissues and neoplasms of mesenchymal, lymphoreticular, or neural crest origin did not contain intracellular keratin. This technic is a useful adjunct for the surgical pathologist in the diagnosis of poorly differentiated neoplasms. Its application either confirmed, modified, or in several instances, changed the original light microscopic impression. The modified or changed diagnoses were confirmed by transmission electron microscopy.
The differentiation of skeletal muscle cells is characterized morphologically by the fusion of myoblasts to form multinucleated muscle fibres. This process takes place gradually during skeletal muscle development in vivo. It can also be followed in tissue culture. Mammalian myoblasts will grow in monolayers, either in primary culture or as established cell lines, and will fuse spontaneously when the culture becomes confluent (for review see Yaffé 1968, Buckingham 1977). The formation of muscle fibres is characterized biochemically by the increased synthesis of contractile proteins (e.g. Devlin and Emerson 1978, Garreis 1979) and their organization into sarcomeric structures (Fischman 1970), by the accumulation of enzymes important in muscle metabolism (e.g. Caravatti et al. 1979), and by the appearance of membrane components such as the acetylcholine receptor (e.g. Merlie et al. 1975), essential for nerve-muscle interaction.
A procedure for dissociating the rabbit aorta into single, functional smooth muscle cells is described. After removal of adventitia and intima, slices of media were incubated with purified collagenase, elastase, and soybean trypsin inhibitor in a Krebs-Ringer buffer modified with Hepes, amino acids, and a [Ca2+] of 0.2 mM. After enzymatic digestion and mechanical shear, the yield of dispersed cells was approximately 25% based on DNA recovered. Greater than 95% of the cells excluded trypan blue and approximately 80-90% adhered to tissue culture dishes. By phase contrast microscopy, most of the cells were elongate and approximately 10 micron X 30 micron in size. The remainder were either spherical or highly crenated and contracted. Electron microscopy of the cells showed that immediately after dissociation greater than 95% could be identified as smooth muscle, though most had undergone some degree of structural change compared to cells in situ. Depending on the preparation, from 5 to 50% of these cells contracted in response to agonists. Cells shortened by 10-15% and developed numerous evaginations when stimulated by angiotensin II norepinephrine, or carbamylcholine. Cells relaxed after washout of agonists and could subsequently be restimulated. Specific inhibitors of each of the agonists blocked the contractile response. Dispersed cells cultured for 1-5 days contracted in even higher numbers than the freshly prepared cells, suggesting restoration of hormone binding and/or contractile function in culture. This preparation provides a system in which the physiology of individual vascular smooth muscle cells may be studied.
The organization of actin and myosin in vascular endothelial cells in situ was studied by immunofluorescence microscopy. Examination of perfusion-fixed, whole mounts of normal mouse and rat descending thoracic aorta revealed the presence of axially oriented stress fibers containing both actin and myosin within the endothelial cells. In both species, the proportion of cells containing stress fibers varied from region to region within the same vessel. Some endothelial cells in mouse mesenteric vein and in rat inferior vena cava also contained stress fibers. Quantitative studies of the proportion of endothelial cells containing stress fibers in the descending thoracic aorta of age-matched normotensive and spontaneously hypertensive rats revealed significant differences. When animals of the same sex of the two strains were compared, the proportion was approximately two times greater in the spontaneously hypertensive rats. The proportion of endothelial cells containing stress fibers was about two times greater in males than in females of both strains. These observations suggest that multiple factors, including anatomical, sex, and hemodynamic differences, influence the organization of the endothelial cell cytoskeleton in situ.
We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.
In order to increase accuracy and precision of quantifying one-dimensional electropherograms by digital signal processing, a set of algorithms is presented that operate on the optical density values acquired by a linear scanning densitometer. These operations include smoothing of the measuring values, background correction by an optimized piecewise linear baseline, and integration of the peak areas after two-sided fit of half-Gaussian profiles to each stained protein band. The algorithms are implemented as BASIC-programs and are readily adaptable to a variety of electrophoretic separations. The procedure has been applied successfully in research [1] as well as in clinical evaluations [2].
This chapter discusses the determination of DNA concentration with diphenylaraine and describes several other color reactions for DNA. The reaction between deoxyribose and diphenylamine is probably the most frequently used color reaction for the determination of DNA. The chapter focuses on the later modifications for the determination of DNA especially in microorganisms and animal tissues and presents the modifications described by Burton, Croft and Lubran, and Giles and Myers. Croft and Lubran were measuring the DNA in saline washes of human stomachs. The material has a high content of sialic acid, which seriously interferes in several color reactions for DNA, including Button's modified diphenylamine reaction. Sialic acid reacts in this method to give a color with a maximum absorption at 550 mμ. Giles and Myers found that the blank readings could be reduced by omitting the sulfuric acid and adding the acetaldehyde to the individual tubes. They also obtained a worthwhile increase of sensitivity by increasing the concentration of diphenylamine and altering the relative volumes of sample and reagent. Giles and Myers encountered turbidities in their reaction mixtures due to impurities in DNA extracts from plant tissues.
Immunofluorescence microscopy using antibodies raised against protein constituents of the different types of intermediate-sized filaments has shown that in HeLa cells filaments containing a prekeratin-like protein (cytokeratin) predominate. The wavy filament bundles decorated by antibodies against prekeratin are similar to those described in other cells of epithelial origin. These bundles of intermediate-sized (6–11 nm) filaments are also described by electron microscopy in intact cells and in cytoskeletal preparations obtained by cell lysis and extraction with low and high salt buffers and Triton X-100. The occasional occurrence of desmosome-attached tonofibrillar bundles of intermediate-sized filaments is also shown. When HeLa cells are treated for long times with colcemid to induce perinuclear whorls of intermediate-sized filaments, these aggregates of filaments are strongly stained by antibody to vimentin, the major polypeptide of the intermediate-sized filaments of murine 3T3 cells. However, they are not stained by antibody to prekeratin, and the display of the prekeratin-containing tonofilament-like structures is similar to that seen in untreated cells. SDS-polyacrylamide gel electrophoresis of cytoskeletons prepared under conditions in which intermediate-sized filaments are retained show the presence of a polypeptide which co-migrates with one component of bovine prekeratin, and a second polypeptide which co-migrates with vimentin purified from mouse 3T3 cells. The data show (i) that two different types of intermediate-sized filaments can be present in the same cell and can be distinguished immunologically; and (ii) that the expression of a prominent epithelial structural marker, i.e. prekeratin-containing filaments, can be maintained in malignancy and continuing proliferation in vitro.
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.
A method is presented for growing large numbers of pure isolated smooth muscle cells from adult human, monkey, and rabbit blood vessels in primary culture.
In the first few days in culture these cells closely resembled those in vivo and could be induced to contract with angiotensin II, noradrenaline and mechanical stimulation. They stained intensely with antibodies against smooth muscle actin and myosin. Fibroblasts and endothelial cells did not stain with these antibodies thereby allowing the purity of each batch of cultures to be monitored. This was consistently found to be better than 99%. The smooth muscle cells modified or “dedifferentiated” after about 9 days in culture to morphologically resemble fibroblasts. At this stage cells could no longer be induced to contract and did not stain with the myosin antibodies. Intense proliferation of these cells soon resulted in a confluent monolayer being formed at which stage some differentiated characteristics returned. The modification or “dedifferentiation” process could be inhibited by the presence of a feeder layer of fibroblasts or endothelial cells, or the addition of cAMP to the culture medium.
Smooth muscle cells which had migrated from explants in primary culture, and cells in subculture, had morphological and functional properties of “dedifferentiated” cells at all times.
The advantages of differentiated rather than “dedifferentiated” smooth muscle cells in culture for the study of mitogenic agents in atherosclerosis is discussed.
With a modified balloon catheter the endothelium of rat thoracic aortae was completely removed to study the interaction between two important responses of the vessel wall to intimal injury: endothelial regeneration and formation of an intimal fibrocellular thickening. Endothelial cells deriving from the uninjured intercostal arteries regenerated by migration followed by proliferation and proceeded as a continuous sheet at a rate of approximately 0.07 mm. per day in the circumferential direction and approximately 6 times faster in the axial direction. Smooth muscle cells appeared in the intima only in areas which were not covered by regenerating endothelium 7 days after injury. The smooth muscle cells formed a multilayered fibrocellular intimal lesion which reached the maximal thickness after 3 weeks. The continuous sheet of regenerating endothelium covered the intimal smooth muscle cells at a slower rate; 6 weeks after injury large areas located most distant from the source of regenerating endothelium still showed modified smooth muscle cells lining the lumen. However, platelets did not adhere to these smooth muscle cells, and the total amount of intimal thickening did not increase between 3 and 6 weeks after injury. We conclude that, in response to intimal injury, endothelial regeneration precedes the accumulation of intimal smooth muscle cells, and that injured intimal areas, which are rapidly covered by continuous endothelium, are protected from the development of a fibrocellular intimal lesion.
The major proteins in isolated synaptic junctions (SJs) and postsynaptic densities (PSDs) have been compared to actin, tubulin, and the major neurofilament (NF) protein by two-dimensional gel electrophoresis and tryptic peptide map analysis. These studies show: (a) tubulin is present in SJ and PSD fractions and is identical to cytoplasmic tubulin, (b) actin in these fractions is very similar to the gamma- and beta-actin found predominantly in nonmuscle cells, and (c) the major PSD protein is distinct from all other known fibrous proteins.
The daily rate of cell replication in the aortic endothelium of the normal rat falls from a maximum of 13% at birth to 0.1-0.3% at age 5-6 months. This residual rate of replication largely represents the turnover rate of the endothelium in normal animals. After renal hypertension of 2-3 weeks duration, however, the rate of replication rises 10-fold to an average value of 1.6%. This increase may represent an increase in turnover; however, it probably represent, at least in part, a proliferative response to cover the expanded luminal surface of the dilated vessel.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Hypertension was produced in male Wistar rats (150 gm. body weight) by complete ligature of the aorta between the renal arteries. Electron microscopic examination revealed that 1 week later the amount of cytoplasmic microfilaments in the endothelial cells of the aortic segment above the coarctation (mean blood pressure 160 mm. Hg) was strikingly increased as compared with normal animals. The endothelial cells in the segment below the coarctation (mean blood pressure 25 mm. Hg) contained few filaments and were similar to the cells in the aortic endothelium in controls (mean blood pressure 105 mm. Hg). Microfilaments measured 40 to 70 angstrom in diameter and were mostly located close to the endothelial clefts, where they formed longitudinal bundles or a network. The bundles of microfilaments contained electron-dense areas similar to the "attachment sites" of the underlying smooth muscle. By using en face preparations of aortic endothelial cells treated with antiactin autoantibodies (AAA) followed by anti-human IgG, it was seen that in hypertensive animals the cells above the ligature were intensely fluorescent when compared with those of the aortic portion below the ligature or those of the controls. The fluorescence was abolished after incubation of the AAA sera with thrombosthenin A. The correlation between electron microscopic and immunologic findings suggests that the microfilaments present in the endothelial cells of hypertensive animals are composed, at least in part, of actin. Endothelial cells so modified may play a role in permeability or may be related to other phenomena such as electrotonic coupling and synchronized contraction of aortic cells during hypertension.
Four-week-old rats received intraperitoneal injections of 3-H-proline, and their thoracic aortas were removed at various times after injection. Autoradiography at the electron microscopic level was carried out on this tissue, together with isolation and chemical analysis of tritiated proline and hydroxyproline from procollagen, and elastin fractions. Autoradiograpic results were expressed in terms of tissue component volumes as calculated by morphometry. Autoradiographic grains were concentrated over medial cells 15 minutes after injection of label. By 30 minutes after injection some extracellular label was present as well; at 3 hours postinjection, large numbers of grains were found overlying cell surfaces, as well as over collagen and elastic fibers. Most grains were present over collagen and elastic tissue 10 hours after injection, although some intracellular labeling persisted. On a grain per unit volume basis, intracellular label steadily decreased over the time interval studied. Collagen showed maximal labeling at 3 hours, but elastic tissue labeling increased up to 10 hours postinjection. Chemical isolation of 3-H-proline and 3-H-hydroxyproline from the three fractions showed that incorporation of label into collagen and elastin is, on a time basis, consistent with autoradiographic labeling and that the labeling products are, in fact, these proteins, to the exclusion of nonspecific labeling.
The tunica media of thoracic aortas from female rats in age from newborn to 12 weeks were analyzed quantitatively by using stereologic techniques in the electron microscope. Collagen, elastin, smooth muscle, myofilaments, Golgi apparatus, ergastoplasm, and surface to volume ratios were among those components quantified. These parameters were correlated with measurements of medial thickness, blood pressure, and tangenital tension in the wall and with biochemical estimates of collagen and elastin. A progressive increase in morphologically recognizable collagen in the media was in parallel with the increasing tangenital wall tension. Biochemical analysis indicated the presence of much soluble collagen in the early stages of development. Elastin was the major component contributing to increasing wall thickness. The elastin laminae were completely formed by 4 weeks of age, but further elastin was laid down as branches extending between medial cells in the aortas of animals between 2 and 12 weeks of age. Smooth muscle cells were ovoid in young animals and contained large amounts of ergastoplasma and Golgi apparatus, but very few myofilaments. With advancing age, these cells became irregular in outline, and their surface to volume ratios doubled. The myofilament volume increased greatly during development, associated with a decrease in "undifferentiated" cytoplasm, ergastoplasm, and Golgi apparatus. The maximal development of the latter two organelles was coincident with large increases in the connective tissue components.
One week after hypertension was produced in male Wistar rats (150 g body weight) by a complete aortic ligature placed between renal arteries, electron microscopic studies showed a striking increase of cytoplasmic microfilaments in the endothelial cells of the aortic segment above coarctation (mean blood pressure 160 mm Hg). These microfilaments measured 40-70 A in diameter and were located particularly close to the endothelial clefts. In "en face" preparation of aortic endothelial cells treated with antiactin autoantibodies (AAA) followed by antihuman IgG, the cells above the ligature of hypertensive animals were intensely fluorescent compared with those of the aortic portion below the ligature or that of controls. The fluorescence was abolished after incubating the AAA-containing sera with thrombosthenin-A, suggesting the presence of actin. There was also an increase transport of horseradish peroxidase and ferritin through the endothelial cell layer. While endothelial cells so modified may play a role in permeability regulation, they may also be related to such mechanisms as electronic coupling and synchronized contraction of aortic cells during hypertension.
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.
Insoluble antigen and antibody derivatives were obtained or antibody. Evidence has been obtained to show that was added to solutions of antigen or antibody. Evidence has been obtained to show that the protein molecules were cross-linked covalently to one another. The immuno-adsorbent properties of these insolubilized proteins were investigated. They were found to be efficient, specific and stable immunoadsorbents, and were used, either in column procedure or in batchwise operation, for the isolation of antigens or antibodies.
Immature cells were identified in all the layers of the wall of the adult sheep carotid. The cells were most numerous in the region of the internal elastic lamina, and showed evidence of a pattern of differentiation which was closely similar to that of developing smooth muscle cells in fetal and neonatal tissues. Lipid droplets and glycogen were frequently present in the immature cells and there was some evidence of their involvement in the formation of elastin. Thickening of the intima was apparent in material from grafted vessels, and the relationship of the immature cells to those present in the intimal thickenings associated with the development of atheroma is discussed.
Sequential changes are described following production of a large area of superficial injury to the internal surface of the aortic wall of the rabbit. Early changes were acute inflammation, intimal thickening and partial reendothelialization of the injured segment. In rapidly reendothelialized regions the subendothelial thickening subsequently disappeared and the gross structure of the injured media was restored.
The human atheroma, the characteristic lesion of atherosclerosis, is an intimal lesion composed of a hard fibrous cap and a soft lipid core. Electron-microscopic studies have in general supported the conclusions of light microscopists: the lipid particles are as widely variable in their ultrastructure as they have been shown to be in histochemical reaction; the fibrous cap is principally collagenous, but contains scattered fibrin strands; tissue necrosis and inflammatory reaction are not present by the usual criteria. The chief result of ultrastructural studies has been the emergence of the smooth muscle cell (myointimal cell) as the principal and essential cell type in atheroma. Fine structural studies of these myointimal cells have, instead of generating clear criteria for their recognition, suggested a spectrum of characteristics ranging from those of a resting connective tissue cell (fibrocyte) to a lipid-laden histiocyte (foam cell); a similarly wide range of function has been implied.
Smooth muscle cells (SMC) of medial explants of swine thoracic aortas appear to undergo in the first few days of culture rapid dedifferentiation toward more primitive forms. The number of cells synthesizing DNA in the explants increases as the number of well differentiated SMC decreases. Electron microscopic autoradiography revealed that more of the cells incorporating 3H-thymidine were poorly differentiated SMC. Thus it appears that in this in vitro situation dedifferentiation of SMC precedes rapid cell proliferation.In early cholesterol-induced atherosclerosis in swine many partially or completely undifferentiated cells are present. The results of the current in vitro study suggest that the undifferentiated cells in the atherosclerotic lesions could arise from mature SMC through dedifferentiation.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
In most cell types intermediate or 10-mm filaments (IF) are a major cytoskeletal organization and, thus, directly or indirectly influence the structural appearance of the cytoplasm. In line with the cell type-specific expression patterns of different IF proteins in normal animal and human tissue, IF typing distinguishes the major tumor groups, as documented by results with several hundred human tumors classified by conventional histologic methods. Carcinomas are characterized by cytokeratins, sarcomas of muscle cells by desmin, nonmuscle sarcomas by vimentin, and gliomas by glial fibrillary acidic protein. Furthermore, certain tumors originating from the sympathetic nervous system, e.g., ganglioneuroblastoma, pheochromocytoma, and at least some neuroblastomas, are characterized by the presence of neurofilaments. Carcinomas can often be further subdivided with regard to their possible derivation by examining their cytokeratin profiles. The IF type characteristic of the cell of origin seems to be kept not only in the primary tumor but usually also in solid metastases. In general, tumors do not acquire additional IF types. Therefore, IF typing can provide an unambiguous and rapid characterization in certain cases, that are difficult to diagnose by conventional techniques. Some useful examples are the small cell tumors of childhood and the discrimination between undifferentiated carcinoma and lymphoma. IF typing of a few tumors has already led to a revision or reconsideration of the original light microscopic diagnosis. The combined results indicate that at least certain carcinomas, as well as certain other tumor types, seem to arise by the selective multiplication of a particular and identifiable cell type present in the normal tissue. The procedure is not restricted to tumor material. IF typing of Mallory bodies, Alzheimer's disease tangles, certain myopathies, and the cells of the amniotic fluid offers further interesting applications. Thus, IF typing should become a valuable new tool both in histology and surgical pathology.
Vimentin-positive, desmin-negative cells were established in culture from the nodule and from apparently normal palmar aponeurosis of a patient with Dupuytren's disease and compared with normal human embryonic and adult fibroblasts or sarcomatous cells. Cells from the nodule display in vitro biological properties that are intermediate between those expressed by normal fibroblasts and sarcoma cells or cells from the nodule transformed with SV40 virus. Thus, they represent an interesting in vitro model of partially transformed human cells. This behavior is not evolutive and justifies the classification of Dupuytren's disease among the benign mesenchymal tumors. The production of high level of plasminogen activator probably explains the local reactive pathology, and could act as a mitogenic stimulus for the proliferation of the nodule itself. Cultures derived from the apparently normal palmar aponeurosis show some but not all the abnormal growth properties of cells from nodules; this may help to explain the onset of local recurrences. Our results suggest that Dupuytren's disease is not strictly local and limited to the nodules, but affects, at least partially, the whole aponeurosis. Dupuytren's nodules could be considered as a model of tumor progression in a benign situation.
Specific antibodies against the intermediate filament protein subunits, desmin and vimentin, were used to characterize the fibroblastic tissue culture cell line BHK21/C13 and the cells comprising baby hamster kidney (BHK). The BHK21/C13 cells have previously been shown to contain desmin and vimentin by biochemical techniques. The results from double immunofluorescence analysis show that both immunologically distinct intermediate filament subunit proteins are expressed simultaneously within the same BHK21/C13 cell, and that the filamentous patterns are very similar, if not superimposable even in cells treated with colchicine. There are some cells that may contain vimentin only. Double immunofluorescence on cryostat sections of BHKs and preparations of dissociated kidney cells demonstrate that the cells, most likely smooth muscle, comprising the blood vessel walls contain vimentin and desmin simultaneously. The simultaneous expression of vimentin and desmin is not a phenomenon which is restricted to tissue culture cells. Thus, the simultaneous presence of these two intermediate filament proteins within the BHK21/C13 cell may not be the result of growth in tissue culture.
Development proceeds by way of a discrete yet overlapping series of biosynthetic and restructuring events that result in the
continued molding of tissues and organs into highly restricted and specialized states required for adult function. Individual
molecules and cells are replaced by molecular and cellular variants, called isoforms; these arise and function during embryonic
development or later life. Isoforms, whether molecular or cellular, have been identified by their structural differences,
which allow separation and characterization of each variant. These isoforms play a central and controlling role in the continued
and dynamic remodeling that takes place during development. Descriptions of the individual phases of the orderly replacement
of one isoform for another provides an experimental context in which the process of development can be better understood.
Fluorescence microscopy with 7-nitrobenz-2-oxa-3-diazole phallacidin was used to survey vertebrate tissues for actin filament bundles comparable to the stress fibers of cultured cells. Such bundles were found only in vascular endothelial cells. Like the stress fibers of cultured cells, these actin filament bundles were stained in a punctate pattern by fluorescent antibodies to both alpha-actinin and myosin. The stress fibers were oriented parallel to the direction of blood flow and were prominent in endothelial cells from regions exposed to high-velocity flow, such as the left ventricle, aortic valve, and aorta. Actin bundles may help the endothelial cell to withstand hemodynamic stress.
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.
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.
Quantitative analysis of proteins separated on polyacrylamide gels requires the use of an accurate, sensitive scanning device. An easily constructed, high-resolution laser beam densitometer is described in this report. The apparatus was built from readily obtainable but precision optical, electronic, and mechanical components. It was designed for and successfully applied to analysis of microslab gels, but also can be used for scanning of cylindrical gels, autoradiographs, and, with simple modification, larger slab gels.
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To study the distribution and the degree of polymerization of actin in regenerating and neoplastic cells we have examined: (1) the immunofluorescent staining of these cells with human antiactin antibodies (AAA), (2) the sensitivity of cellular actin to AAA staining after treatment with the actin-depolymerizing factor present in plasma or serum of several animal species, and (3) the total and relative amounts of F- and G-actin in tissue preparations, determined by means of differential centrifugation and densitometric analysis of stained sodium dodecyl sulfate-polyacrylamide gels. There was little or no difference in staining intensity between normal and regenerating or tumoral tissues when treated with AAA, but AAA staining of normal tissues was abolished, whereas staining of regenerating and tumoral tissues remained intense after incubation with actin-depolymerizing factor. Gel analysis showed that normal, regenerating, and neoplastic epidermal cells contained similar amounts of actin. Compared to controls, regenerating liver tissue contained slightly more total actin, nearly the same amount of G-actin, but a substantially increased amount of F-actin. These results suggest that: (1) compared to controls, a greater proportion of regenerating and neoplastic tissue actin is stabilized against the action of actin-depolymerizing factor; (2) changes involving actin in regenerating and neoplastic epithelial cells reflect changes in its degree of polymerization rather than its total amount.