DISTRIBUTION OF VIMENTIN AND DESMIN ... - Science Direct

CopyrIght @ 1982 hy Academic Pres. Inc. All rightc of reproductwn in any form reserved (K114-4X27/R?i020329-(~,%02 00/O

Experimental

DISTRIBUTION IN SMOOTH

Cell Research 137 (1982) 329-340

OF VIMENTIN MUSCLE

AND DESMIN TISSUE

AND AVIAN

FILAMENTS

OF MAMMALIAN

AORTA

ERIKA SCHMID,’ MARY OSBORN,’ ELISABETH RUNGGER-BRANDLE,” GIULIO GABBIANI? KLAUS WEBERZ and WERNER W. FRANKE’ ‘Division of Membrane Biology and Biochemistry, Institute of Cell and Tumor Biology, German Cancer Research Center, D-4900 Heidelberg, ?Max-Planck Institute for Biophysical Chemistry, D-3400 Giittingen, Federal Republic of Germany, and 3Department of Pathology, University of Geneva, CH-1211 Geneva 4, Switzerland

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

Intermediate-sized (7-l 1 nm) filaments are a widespread class of fibrils with similar morphology which can be formed by biochemically and immunologically distinct proteins ([l-3]; for review see [4]). Among mesenchymally derived cells, certain cell types contain only intermediate-size tilaments of the vimentin type [3, 51, whereas others, specifically those of myogenic origin, produce filaments containing a different protein called desmin or skeletin [68]. Both proteins, vimentin and desmin, differ somewhat in apparent molecular weight and isoelectric point but share cer-

tain biochemical properties such as low solubility, a similar pattern of phosphorylated variants [lo, 111, and some related cleavage fragments obtained by enzymatic proteolysis or chemical cleavage [lo, 1214]. Such biochemical similarities are recognized even in vimentin and desmin from species as taxonomically distant as chicken, rodents and man ([lo]; for amino acid sequence similarities between desmins from avian and mammalian species, see [15]). Both vimentin and desmin filaments are ’ To whom offprint requests should be directed

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known to form whorl-like perinuclear ag- adventiria layers and analysed. The samples were directly extracted in the specific sample buffer gregates when cells are treated with cer- either or frozen. Human aortae were obtained at autopsies between tain antimitotic drugs [ 1-3, 161.In addition, 2.5 and 20 h post mortem. filaments of both the vimentin and desmin For comparison, myometrial tissue from porcine and type can occur within the same cell, in human uterus and chicken gizzard muscle were obas described (cf r21, 241). Cultured rat vascuparticular during myotube formation in tained lar smooth muscle-derived cells (RVF-SMC) and culvitro [ 17, 181 and in some cultured cell tured cells from embryonal leg muscle of chicken were lines of unknown embryonal cell origin grown as described [25, 261. [lo, 131. The co-existence of vimentin and Preparation of cytoskeletons desmin in Z-discs of developing and mature Since proteolytic degradation has been found to be cross-striated muscle is a matter of debate a major problem during analysis of vascular tissue proteins, especially in preparations of cytoskeletons [l, 2, 17-191. [21], various procedures were used to try to miniRecently, vimentin has also been ob- mize proteolysis. In some experiments, the tissue was minced into small pieces and incubated before homoserved in certain smooth muscle cells, i.e., genization with 2 mM di-iso-propyl-fluoro-phosphate smooth muscle cells of the vascular sys- (DFP, Fluka, Buchs, Switzerland) in TBS (10 mM Tris-HCl, pH 7.4, 140 mM NaCl) for 15 min at room tem [20, 211. Moreover, differences be- temperature. Procedures for cytoskeletal preparations tween different species have been noted as were essentially as described (1211,cf 1271).Briefly, tissue samples -were homogenized-in TBS-in a Polyto the simultaneous occurrence of desmin tron homogenizer (15-30 set, setting 3) and briefly extracted in TBS containing 1% Triton X-100. All in the same vascular smooth muscle tissue extraction and washing steps were carried out in the [21]. We have therefore examined the pres- presence of 0.4 mM protease inhibitor (phenyl-methylence of both filament proteins, vimentin sulfonyl-chloride, PMSF) and 5 mM EDTA or 5 mM EGTA. Insoluble material was collected at 3 500 g for and desmin, in aortae of phylogenetically 15 min. resusuended in TBS containing 0.6 M Kl. distant vertebrate species by gel electro- 0.5 % Triton X-100 and stirred for 20 min (4°C). After centrifugation, the pellet was extracted in TBS conphoretic analysis and immunological tech- taining 1.5 M KCl, 0.5% Triton X-100 for 30 min niques. We report here that in diverse (4°C). Finally, the insoluble residue was washed in 10 mM Tris-HCl (pH 7.4). vertebrates, including chicken, rat, cow and man, cells containing vimentin occur in all Gel electrophoresis layers of the wall of the larger arteries, For one-dimensional polyacrylamide gel electrophoreincluding the smooth muscle cells of the sis in the presence of SDS the system of Laemmli [28] was used. For two-dimensional gel electrophoretunica media. In addition, we show that sis [29], the tissue was either directly solubilized in some smooth muscle cells occurring in the the specific lysis buffer [29] or was pre-treated with low concentrations of SDS and 2-mercaptoethanol as aorta also contain desmin filaments but that described [30] or was boiled for 5 min in 10 mM sodium-potassium-phosphate buffer (pH 7.5) containthe ratio of vimentin and desmin can differ ing 5% SDS and 10% 2-mercaptoethanol. After cenbetween different species. trifugation, proteins solubilized were precipitated with acetone at -20°C for 3 h, collected by centrifugation, and the pellet was dried and used for isoelectric focusing.

MATERIALS

AND METHODS

Tissues Aortic and heart tissues of man, cow, rat, and chicken were used. For immunofluorescence microscopy, whole pieces of tissues were directly frozen at - 150°C in isopentane [22, 231. For gel electrophoretic analysis, pieces of whole aortae and of large arteries were used. In addition, in the case of man and cow tunicn media segments were stripped from the intima and E.rp

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Antibodies Prenaration and characterization of guinea uin antibodies raised against murine and human vimentm have been described l3.251. In addition, vimentin antibodies that were aftin& purified on vimentin from rabbit chondrocytes were used. Two different antibody preparations against chicken gizzard desmin were used. The preparation of guinea pig antibodies as used, for example, for figs 5b and 7b has been previously described [24]. The rabbit desmin antibody is a new

Distribution of vimentin and desmin filaments in smooth muscle tissue

331

preparation prepared against highly purified desmin from chicken gizzard (cf [ 151)and was affinity-purified on desmin prepared from the same source. It stained Z-lines of cross-striated muscles and, in addition, intercalated discs of cardiac muscle of avian and mammalian species. A characterization of this antibody will be presented elsewhere [31].

Zmmunojluorescence microscopy Procedures for indirect immunofluorescence microscopy on frozen sections were as described (cf [23, 241). Double immunofluorescence microscopy was performed as described for cultured cells [32].

RESULTS Gel electrophoresis In comparative studies on aortic tissue from diverse species it has become clear that this tissue can exhibit high proteolytic activity. Degradation by an endogenous protease can result in the loss of a high proportion, and sometimes of all, of the intermediate filament protein identified by two-dimensional gel electrophoresis using the O’Farrell[29] procedure for sample lysis. A characteristic series of degradation products resulting from the activity of an endogenous Ca2+-protease known to digest vimentin as well as desmin has been described [ 10, 21, 331. Especially strong protein breakdown has been observed when vascular tissues were not denatured immediately upon removal from the animal, and this is a special problem when tissue samples are collected

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Fig. 1. (a) SDS-polyacrylamide and (b, c) two-dimensional (IEF, direction of isoelectric focusing; SDS, second dimension in presence of SDS) gel electrophoresis showing species differences of mammalian and avian vimentin in cytoskeletal preparations from RVF-SM cells (u, slot I, and b), from cultured cells of chick embryo leg muscle (a, slot 3) and by coelectrophoresis of both ((I, slot 2, andc). Avian desmin (chicken gizzard cytoskeleton) is shown for comparison (a, slot 4, horizontal bar). Note the difference in electrophoretic mobility and isoelectric pH between mammalian (arrowheads in CI, V, in h and c) and chicken (arrows in a, Vr in L.) vimentin. Dots denote the position of actin. Erp

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---

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Fig. 2. SDS-polyacrylamide gel electrophoresis of cytoskeletal preparations of aortic tissues from different vertebrate species showing the presence of considerable amounts of vimentin (arrowheads) in man (n. slot l), chicken (a, slot 2), cow (a, slot 3, and b, slot I) and rat (b, slot 2). Note that the ratio of vimentin to desmin differs greatly, e.g., in bovine (b, slot 1) and rat (h, slot 2) aorta (desmin is indicated by horizontal bars). Dots denote actin.

from human autopsies or from animals killed at the slaughterhouse (data not shown). Therefore, for one- and twodimensional gel electrophoretic separations, samples have been directly lysed in the SDS-containing lysis buffer of Laemmli [28] and boiled. The solubilized proteins have been precipitated with acetone and transferred into the urea-containing buffer Exp Cd RPS 137 11982)

used for isoelectric focusing (see Materials and Methods). Vimentin from mammalian and avian species shows a slight difference in mobility on one-dimensional (fig. la) and twodimensional (fig. 1b, c) gel electrophoresis. Likewise, desmins from smooth muscle tissues of mammalian and avian species show subtle differences in mobility on two-dimensional gel electrophoresis as first described by Lazarides & Balzer [34]. In addition, differences in tryptic peptide Iingerprints and in partial amino acid sequences have been documented for mammalian and avian desmin [IS, 351. These observations indicative of changes of these molecules during vertebrate evolution make it necessary to examine the identity of vimentin and desmin from vascular tissues by co-electrophoresis with authentic vimentin or desmin from the same species, or at least from the same group of vertebrates. Fig. 2 shows a one-dimensional comparison of aortic cytoskeletal proteins from man, chicken, cow and rat, and fig. 3 the two-dimensional comparison from the same species. In man, rat and chicken, vimentin is the predominant intermediate filament protein (cf [20, 211). Only in preparations of thoracic aorta from the cow we have been able to find a higher proportion of desmin, and this is especially pronounced in cytoskeletal preparations made from dissected and stripped tunica media tissue (figs 2a, slot 3, 2b, slot 1, and 3b). In preparations of whole proteins and cytoskeletal residue proteins from human aorta, including preparations of stripped medial smooth muscle tissue, we have failed to detect desmin (fig. 2a, slot 1, fig. 3~). The specific identities of avian and mammalian vimentins and desmins have also been demonstrated by immunological cross-reaction, after separation by gel electrophoresis and


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IEF v l-l

SDS

a

b

I

c

D

nv

“’

d

Fig. 3. Two-dimensional gel electrophoresis of cytoskeletal preparations of aortic wall proteins from different species: (a) man (stripped tunica media); (b) cow (stripped tunica media); (c) rat (whole tissue);

(d) chicken (whole tissue). Vimentin (V) is a major protein in all species whereas desmin (D) is clearly detected only in rat (c), cow (b) and chicken (d). A, residual actin.

blotting on nitrocellulose paper, using guinea pig antibodies to murine or human vimentin (data not shown).

well known from electron microscopic studies [36]. The results with vimentin antibodies show that the vast majority, and in some species perhaps all, of cells present in different regions of the aorta are stained, including endothelium (for occurrence of vimentin in endothelial cells see also [27, 371). These observations confirm and extend the recent reports of Frank & Warren [20] and Gabbiani et al. [21]. In contrast to these reports our results show that desmin-positive cells are also found in aortic

Immunolocalization The localization of vimentin and desmin in sections through frozen tissues of different species, i.e. man, cow, chicken, rat, by immunofluorescence microscopy using antibodies to vimentin and to desmin is shown in figs 4-7. These micrographs stress the existence of different layers of aortic tissues 22-811803

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aorta we have also observed, in outer regions of the tunica media, bundles of densely packed, mostly longitudinally oriented smooth muscle cells strongly stained with antibodies to desmin (fig. Sf) but not with antibodies to vimentin (fig. 5e). By contrast, cells interspersed between these longitudinal smooth muscle bundles are stained with antibodies to vimentin, but not with desmin antibodies (fig. 5 e, f). Using the new rabbit antibody preparation against desmin we have now also been able to stain a number of cells present in the Fig. 4. Immunofluorescence microscopy on frozen tunica media of human thoracic aorta, sections of rat aorta after staining with antibodies to (a) vimentin; (b) desmin. Note sparsity of desmin- whereas only sparse distributions of despositive cells in outer part of tunicu media (b, bot- min-positive cells are seen in the t. intima tom) using affinity-purified rabbit antibodies against desmin (6). L, lumen. Bar, 30 pm. (fig. 6a, b). Thus, the desmin localization now observed differs from previous results [21] which have failed to detect this tilament protein in the human tissue. Similarly, large human arteries such as heart coronary tissue from all species examined. However, the number of such cells and their location arteries present vimentin staining in nearly within the aortic wall can vary with the spe- all cells of the t. media (fig. 6c), but also groups of smooth musc!e cells that stained cies and the position along the aorta. In rat thoracic and abdominal aorta des- with the new desmin antibodies (fig. 6d). min-positive cells are found in both tunica intima and t. media and appear to be rela- Fig. 5. lmmunofluorescence microscopy of frozen sectively more frequent in the inner layers of tions from cow aorta, presenting the reaction with antiagainst vimentin (a. c, e) and desmin (6, d, f). the t. media (fig. 4b). In bovine aorta (fig. bodies The difference of the pattern of distribution of vimen5) cells stained with desmin antibodies are tin and desmin-positive cells is demonstrated by (c, d), using simultaneous insparse in the t. intima and in the innermost double-immunolabeling cubation of guinea pig antibodies against vimentin and layers of the t. media (fig. 5b, d). In the rabbit antibodies against desmin, followed by staining a mixture of fluorescein-conjugated (FITC) anticentral regions of the t. media which are with rabbit IgG (desmin, d) and rhodamine-conjugated antiknown to contain only smooth muscle cells guinea pig IgG (vimentin, c). The tunicu intima (I in u) shows a high density of vimentin-positive cells [36, 381, desmin-positive cells are prefer- ((I, c), including endothelial cells (E), but only sparse entially arranged in circular or helical and scattered (b. guinea pig antibodies) or no (4 that are stained with desmin antibodies. Most of smooth muscle tracts (fig. 5 b, d) separated cells the tunica media (M) contains tracts of cells stained by other smooth muscle cells that seem with both antibodies (u-d) and interspersed cells stained only by vimentin antibodies but not antibodies positive for vimentin only. Double antibody to desmin (u-d). In outer regions of tunicu media labelling (fig. 5c, d) shows that cells posi- bundles of more or less longitudinally oriented distinct of closely packed smooth muscle cells are tive for desmin appear also positive for bundles seen which are not significantly stained with vimentin vimentin, indicative of the co-existence of antibodies (e) but are strongly stained with desmin cf). Note intense staining of vimentin antiboth types of intermediate filaments in this antibodies bodies in cells interspersed between these bundles type of smooth muscle cells. In bovine (e). L. lumen. Bar, 30 pm. E.rp Cd

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Fig. 6. Immunofluorescence microscopy on frozen sections of (a. b) human aorta and (c, d) human coronary artery after reaction with antibodies against vimentin (a, c) and desmin (b, d; affinity-purified antibodies from rabbit). Note staining of most, probably all. cells with vimentin antibodies in both human

arteries (a, c), whereas fewer cells (b), often occurring in groups Cd), of the tunica media are stained by desmin antibodies. The wavy lines in the left part of (d) are elastic fibers which can show unspecific fluorescence. L, lumen. Bar, 50 pm.

Clearly, the number of vimentin-positive cells present in this tissue exceeds the number of cells decorated with desmin antibodies (fig. 6c, d). The anatomy of the avian aorta differs profoundly from that of the mammalian aorta. Here the t. media shows rather regularly alternating layers of tracts of typical smooth muscle cells separated by layers of fibroblastoidal “interlamellar connective

Fig. 7. immunofluorescence microscopy on cryostat sections through aortic walls of chicken after staining with antibodies against vimentin (a, c) and desmin (b, guinea pig antibodies; d, rabbit antibodies). Note staining of most, probably all, cells with vimentin antibodies, whereas only the typical tracts of smooth muscle cells in the tunic-a media are strongly stained by desmin antibodies (b, d). This difference of distribution in the staining pattern obtained by these both antibodies is demonstrated by double-labeling in (c) (vimentin, rhodamine) and (d) (desmin, FITC). Antibody preparations and incubations are the same as described in fig. 5. Fig. 7b shows the staining of antidesmin serum from guinea pig, not affinity purified. L, lumen. Bar, 30 Km.

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Distribution of vimentin and desminfiiaments in smooth muscle tissue

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tissue cells” [38, 391. After staining with tive cells in general correspond to the vimentin antibodies practically all cells of sparse and loosely arranged smooth muscle all layers of the chick aortic wall are posi- cells of the tunica intima and the concentritive (fig. 7a, c). However, when frozen sec- cally arranged tracts of smooth muscle cells tions of chicken aorta are examined with characteristic of the tunica media [36]. antibodies to desmin only the more or less Groups of vascular cells positive for desmin circularly arranged tracts of smooth muscle but not for vimentin have so far only been cells are positively stained (fig. 7h, d). detected with certainty in outer regions of Double immunolabelling of the same sec- the bovine aorta where they are arranged in special longitudinal smooth muscle buntion of chicken aorta with both vimentin and desmin antibodies is shown in fig. dles. Since all cells present in the tunica 7c, d. media of mammalian blood vessels have the typical morphology of smooth muscle cells (e.g., [36, 38-j)our observations suggest that DISCUSSION two types of muscle cell can occur in this Abundance of intermediate-sized filaments tissue, viz. cells containing only vimentin in vascular tissue, including aortic walls, filaments and other cells in which both has been noted in electron microscopic vimentin and desmin filaments appear to studies [41-431. Our observations indicate coexist. Whether these different smooth that most cells of the aortic wall, including muscle cell types have different functions smooth muscle cells, contain vimentin [3, or represent different routes of myogenic 20, 211, in agreement with the origin of differentiation remains to be examined. vascular tissues from embryonal mesenThe situation is different in the avian thyme. However, whereas the endothelial aorta. Here, layers of cells with typical cells contain exclusively intermediate-sized smooth muscle morphology [39, 401 are filaments of the vimentin type [3, 27, 371 strongly positive for desmin and may also the situation appears to be more complex contain vimentin (for occurrence of desmin and heterogeneous in deeper layers of the in chicken vascular cells see also ref. [7]). vascular wall, including the tunica media. The layers of cells with branched processes In addition, interspecies differences with and fibroblast-like morphology (“interlarespect to the distribution of vimentin and mellar connective tissue cells”) sandwiched desmin in vascular cells have been noted. between the smooth muscle cell tracts conIn contrast to recent reports [20, 211 we tain only vimentin filaments and are negashow in this study that desmin occurs in tive for desmin. some cells of vascular tissue and that three The gel electrophoretic identification of different types of cells can be distinguished intermediate filament proteins, notably desby immunofluorescence microscopy using min, in vascular tissues seems to present, new antibodies to desmin: (i) cells con- at least in some species, a preparative taining only vimentin filaments; (ii) cells problem. Recently, Johnson & Yun [42] which seem positive for both vimentin and have described a preparation of intermedesmin; and (iii) cells only stained by des- diate filament protein of approximate M, min antibodies (for demonstration of cul- 5.5000 from bovine pulmonary arteries, but tured vascular cells stained with antibodies their data did not allow distinction between to desmin see also ref. [44]). Desmin posi- vimentin and desmin. In the present study Elrp Cd RCF 137 llY82)

Distribution of vimentin and desmin filaments in smooth muscle tissue we have shown the presence of desmin in aortic tissue from rat, cow and chicken, but not in human autopsy material. It may be that regional differences and/or proteolytic degradation of desmin have contributed to previous gel electrophoretic failures to identify desmin in vascular tissue from several species [21] as well as to our failure in the present study to detect desmin in extracts from human vascular tissue (e.g., figs 2 and 3a). Co-existence of two different types of intermediate-sized filaments in the same cell is a relatively unfrequent phenomenon in tissues. Whereas the occurrence of both vimentin and cytokeratin filaments is common for many cultured epithelial cells [3, 25, 321 and desmin filaments have been described to coexist with vimentin filaments in certain cultured embryonal cells of unknown cellular origin [l, 2, 10, 131, only glial filaments have been reported to coexist with vimentin filaments in cells grown in situ, i.e. nervous tissue ([45, 461; for occurrence of both glial filaments and vimentin filaments in cultured astrocytes and glioma see [45-481). The apparent co-existence of relatively large amounts of both vimentin and desmin filaments in certain vascular smooth muscle cells indicates that these two different classes of intermediatesized filaments can also occur in a functional muscle tissue (the possibility that a very low proportion of vimentin might also occur in chicken gizzard has recently been discussed, though not demonstrated, in ref. [33]). Thus, the observations reported in the present study suggest that the existence of large amounts of vimentin filaments is fully compatible with the contractile function of terminally differentiated smooth muscle cells of the vascular system. The identification of different types of cells of the vascular wall by their different

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contents of intermediate-sized filaments should be helpful in the characterization of the specific types of cells involved in the formation of the vascular system. In view of the common origin of all cells of the blood vessel system from early embryonal mesenthyme it would also be important to know when during angiogenesis and vascular differentiation, the various cell types of the vascular wall appear and what the sequence of synthesis of vimentin and desmin filaments is in the different vascular cells. The authors thank C. Grund and S. Schiller for expert assistance with the light and electron microscopy, and Dr J. Caselitz for discussion. This work was supported in part by the Deutsche Forschungsgemeinschaft and the Swiss National Science Foundation.

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Received June 2, 1981 Revised version received August 31, 1981 Accepted September 7, 1981

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