Vimentin filaments in cultured endothelial cells form butyrate-sensitive juxtanuclear masses after repeated subculture

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Experimental

VIMENTIN

Cell Research 138 (1982) 159-166

FILAMENTS

IN CULTURED

FORM BUTYRATE-SENSITIVE MASSES

AFTER

M. HORMIA,’

REPEATED

E. LINDER,’ R. A. BADLEY’

ENDOTHELIAL

CELLS

JUXTANUCLEAR SUBCULTURE

V.-P. LEHTO,’ T. VARTI0,“3 and I. VIRTANEN’

‘1)epartments of Bacteriology and Immunology, 2Pathology, and 3Viroioxy, SF-00290 Helsinki 29, Finland, and ‘Unilever Research, Colworth Laboratory,

University of Helsinki, Sharnbrook, Bedford, UK

SUMMARY

In primary cultures of human umbilical vein endothelial cells vimentin-suecific fluorescence was seen in spreading cells as distinct juxtanuclear aggregates and in full; spread cells as typical perinuclear coils and cytoplasmic fibrils. After repeated subculture the cells increased in size and showed vimentin-specific-staining as juxtanuclear cap-like aggregates also in fully spread cells. Treatment of late-passage cultures with sodium butyrate led to flattening of the cells and reappearance of fibrillar vimentin organization. Treatment of the late-passage cells with anti-mitotic drugs brought about the formation of solitary juxtanuclear masses of vimentin. In primary cultures, an occasional coalignment of microtubules and vimentin filaments was seen in double IIF, but in late-passage ceils only after culture in the presence of sodium butyrate. Similarly, centrioles could be shown to be close to the juxtanuclear vimentin aggregates. The results indicate that vimentin filaments in cultured umbilical vein endothelial cells have a spontaneous tendency to aggregate independently of the organization of microtubulcs.

Cultured venous endothelial cells appear to contain only vimentin-type of intermediate filaments [ 11, often spontaneously concentrated around the nucleus forming typical perinuclear filament coils [l-5]. Vimentin filaments in endothelial cells can be typically rearranged by treating the cells with antimitotic drugs [l], and even during mitosis they persist in endothelial cells as a closed perinuclear ring until it is cleaved into two symmetrical crescents at late telophase [3]. In previous studies on intermediate filaments of cultured endothelial cells either primary cultures or early passage cells have been used [l-6]. Since both nutritional conditions and culture age have a profound influence on the metabolic events and morphologic characteristics of endothelial cells 11-811804

[7-lo] it is conceivable that also cytoskeletal elements could be affected by culture conditions. Here we show that repeated subculture of human umbilical vein endothelial cells in the presence of fibroblast growth factor (FGF), leads to a microtubulus-independent aggregation of vimentin filaments into juxtanuclear masses that can be rapidly respread with sodium butyrate. MATERIALS Cell culture

AND METHODS

and cytoskeletal

preparations Human umbilical cords, obtained at normal deliveries at the Department of Obstetrics and Gynaecoiogy, University Central Hospital of Helsinki were used as a source of endothelial cells. The cells were har-

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ef al.

Vimentifz filuments in endothelitil ceLls vested from the umbilical veins essentially as described by Jaffe et al. [If, 121 by coflaaenase nerfusion (type CLS II, Worihingtoi, Freehold)). The Eeffs were cultured on plastic Petri dishes in HAM’s FIO medium (Gibfo Biocult, Glasgow) supplemented with 20% pooled human AB serum (Finnish Red Cross Blood Transfusion Service, Helsinki), IO0 ngfml fibroblast growth factor (FGF, Coftaborativc Research, Cambridge) and antibiotics. The culture medium was changed every two days. The confluent cells were subcultured by standard trypsinization. Throughout the culture period antibodies against clotting factor VIII showed a granular cgtopfasmic fluorescence in indirect immunoffuorescence microscopy [ 121. For some experiments the cultures were exposed to demecolcine (Cofcemid@, Ciba, Milan), at 5 pg/ml for 4 h or to sodium butyrate (3 mM; Merck AG, Darmstadt) for 5 days. For metabolic tabelling experiments confluent cultures were incubated with [“Hlfeucine (IO pCi/mf; 20 Ci/mmof; Radiochemical Centre, Amersham) in a feucine-free medium for 48 h. Adherent cytoskefetons of cultured endothefiaf celfs were produced as described earlier for cultured fibroblasts [13]. Briefly, the cells were first extracted with 0.5 % Triton X-100 in 50 mM Tris-HCI, pH 7.2, suppiemented with 1 mM phenylmethyl sulfonyffluo~de (PMSF) at 0°C for 30 min. Thereafter the cells were extracted with a low and a high ionic actomyosin extraction buffer as described earlier in detail [ 13, 141.

Indirect immunofluorescence microscopy Rabbit and guinea pig antibodies against vimenrin, isolated From cytoskeletons of cultured human fibrobfasts using the preparative gel efectrophoretic technique were used as described elsewhere

Antibodies.

Fig. 1. Distribution of vimentin and tubulin in endotheliaf cells at different stages of culture. Confluent primary cultures show typicaffy perinucfear coils or rings of vimentin-snecific fluorescence (Al. A tine fibriilar cytopfasmic‘fluorescence is seen’ in cells from the 6th passage (B). In endothefial cells from the 12th passage-vimentin-s’pcific fluorescence is often located to spherical phase-dense bodies (arrows in C, U). In binucfeate cells, often found in such cultures the viment~n aggregates are consistently seen between the two nuclei (inserts in C, D). In double IIF of endothefial cells at the 12th passage (E, F) the mass-like juxtanuclear staining is seen with anti-vimentin antibodies (E) whereas tubufin-specific fluorescence is located to cytoplasmic fibrils (insert in F) or to centriofes located close to the vimentin masses (arrows in F). In these cultures a fibrillar tubufin-specific fluorescence could fx obtained at different levels of focus (compare ceils in F, asterisk in a cell visuafized at a different level of focus in the insert). In primary cultures of endothelial cells a dense tubulin-specific cytopiasmic fibriffar fluorescence is seen (I?), whereas a more distinct fibrillar fluorescence is seen in subcultured cells (8th passage) with a more flattened ag pearance (H). Note the distinct centriolar fluorescence in subcultured cells (arrows in F and N). (A-D)

X300; {E, F, H) X600; (G) x700.

161

in detail [2]. Both antibodies gave a singfe line of reaction on eiectrophoretically separated fibrobfast nolvnedides using the immunoblottirur. technicrue. ?ub;fin antibodies-were raised in rabbits>gainst r
Polyacrylnmide gel electrophoresis Polyacrylamide gel electrophoresis in the presence of sodium dodecyf sulphate (SDS) was done according to Laemmli f16] using g% slab gels. Radioactive gels were processed for ~uoro~~hy as described by Bonner & Laskey C171.For the immunobio~ng technique the method bf Towbin et al. fig} was us;d. BMy, efectrophoreticalfy separated pofypeptides were transferred onto a nitroceflufose sheet (Millipore@, Bedford) using a commercial destaining apparatus (Pbarmacia, Uppsafa). Amido black (0. I %) was used for nrotein staining. For rabbit antibodies, the nitrocefluiose sheets were first allowed to react with ,rabbit anti-vimentin antibodies ff : 100 in NaCf-P buffer SUDplemented with 3% bovine serum albumin and 10% normal swine serum). Thereafter the sheets were washed, expose& to swine anti-rabbit IgG antiserum and after washing to rabbit peroxidase-anti-peroxidase complex.

RESULTS Intermediutefiluments and microtubules in e~d~theli~l celis In well-spread, subcon~uent primary cuftures vimentin filaments formed, a cytoplasmic network with a tendency to perinuclear concentration and in confluent primary cultures most of the cells showed perinuclear

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Vimentin Jilaments in endotheliul cells

I63

rings or coils of vimentin filaments (fig. IA). Subcultured endothelial cells were clearly increased in size after several passages and expressed a fibrillar vimentin-specific staining (fig. IB). In cultures passaged repeatedly in the presence of FGF (from 10th up to 18th passage) ca 30% of the cells showed a distinct aggregation of vimentin into phase-dense juxtanuclear masses (fig. 1C, U). In such cells the cellular periphery appeared to lack fibrillar vimentin. In binucleate cells, often found in late-passage cultures. one spherical aggregate of vimentin was usually seen either between the nuclei or in the medial plane of the cell adjacent to the two nuclei (fig. 1C, I), inserts). In contrast to vimentin filaments, the organization of the cytoplasmic microtubu-

lar complex did not seem to be dependent on the cell passage number. A fine fibrillar network of microtubules could be decorated in endothelial cells throughout the culture period (fig. IF insert, C, I?). The centrioles, decorated as bright doublets of spots with tubulin antibodies, were apparently not included in the vimentin aggregates but were located adjacent to the vimentin mass (fig. lE, F). In spreading endothelial cultures both cells from early and late passages showed a juxtanuclear phase-dense aggregate of vimentin (fig. 2A, B, E, F) during the first 60 min. Celfs from early passages showed a fibrillar vimentin organization already after 2 h in culture (fig. 2C, II) whereas in late-passage cells the vimentin aggregates appeared to persist throughout the whole spreading period (fig. 2G, H).

Fig. 2. Distribution of vimentin in spreading (A-H), colcemid-treated (I, .I) and butyrate-treated (K, L, IV) endothelial cells from orimarv cultures (A-D I and K) and from the 12th p&sage ~l&Y, I, L &). Freshly seeded endothelial cells both in urimarv culture and in early passages show a phase-dense (ariow in B) juxtanuclear aggregate brightly decorated with vimentin antibodies in iIF (A) during the first 30-60 min of spreading. Note the bright fibril (arrow in A) extending to the nuclear periphery from the aggregate. A fibrillar vimentin-specific fluorescence is seen in spreading cells already after 2 h in culture (C, D). In spreading cells from cultures passaged over 10times vi~ntin-s~ci~c fluorescence is seen as chase-dense juxtanuclear masses both after 1 h (E, ‘F) and 2 h (G, H) in culture. Note the fine vimentin fibrils extending in E, G to the nuclear periphery (arrows). Demecolcinc (5 g/ml for 4 h) causes a typical coiling cytoplasmic vimentin-specific fluorescence in cells from a primary culture (I), whereas a distinctly different cap-like staining is seen in subcultured cells (f). After sodium butyrate (3 mM for 5 days) treatment both cells in nrimarv culture (comoare K to A in fig. 1) and subc;lturedWcells (cornpar; L to ,!.I in fig. 1) are distinctly flattened and show a fine fibrillar cytoplasmic vim&n-specific staining (K, L). Typical perinuclear filament coils can still be seen in EC(arrows). Vimentin aggregates arc not seen in butyratetreated subcultured cells (L). In double IIP with tubulin (M) and vimentin (IV) antibodies an occasional coalignment of the fluorescent fibrils is seen in endothelial cells s&cultured 12 times and exposed to sodium butyrate (arrows in M. iv). (A-f) x400; (J) x300; (EC)x700; (L-N) x400.

Treatmsnt of thr endotheiial cells with de~~e~~lcine or ~~dilirn but~rate When primary cultures of endothelial cells were treated with demecolcine (5 pg/ml for 4 h), vimentin filaments were rapidly rearranged into perinuclear coiling bundles (fig. 21). After a similar treatment, cells from late passages (10-18) showed solitary juxtanuclear vimentin masses and filament coils could. only occasionally be found (fig. 2.J). Addition of sodium butyrate to both primary and late passage cultures caused an obvious fattening of the cells (fig, 2K, 15). In treated primary cultures vimentin-specific fluorescence was seen both as cytoplasmic fibrils and as perinuclear coils (fig. 2K). On the other hand, in late passage cells a fine tibrillar vimentin-specific ffuorescence could be seen and cells with juxtanuclear vimentin aggregates were not found (fig. 2L). In such cells an occasional coalignment of microtubules and vimentin Lip C’d Rcs 13x (19X‘?,

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Fig. 3. Analysis of polypeptides in cytoskeletal prepa-

rations of umbilical vein endothelial cells subcultured uo to 6 times. A number of polypeptides is seen in whole cells both by Coomass‘ie biuh staining and by ~uorogr~phy of metabolically labelfed cells (lane I). Instead, only a major polypeptide with an apparent MW of 58 kD (arrowhead) is seen in the cvtoskeletal preparations b&h after C&massie blue staining (lane 2) and after fluorography (lane 3) together with some polypeptides of higher and lower MW. The 43 kD &lye&tide remaining in the cytoskeletons comigrated with purified actin (lane 4) and the 58 kD polypeptide with purified vimentin (lane 5). Using the immunoblotting technique the 58 kD polypeptide could be identified as vimentin on electrophoretically separated cytoskeletal oolypentides from endothelial cells transfeired onto anit&cdllulose sheet and processed using the PAP-technique (lane 6). MW markers are indicated on the left hand side.

filaments could be seen in double IIF (fig. 2M, N). Cytoskeletal preparations of endothelial cells Treatment of primary cultures of endothelial cells with Triton X-100 followed by low and high ionic buffers led to a disappearance of most of the cytoplasmic contents. The cells from both primary cultures or from late passages remained on the growth substratum as distinct ghosts with nuclear residues and showed a bright fibrilExp Ceil Res 138 (1982)

lar vimentin specific fluorescence in IIF (results not shown). In polyacrylamide gel eiectrophoresis of whole cells from both early and late passages numerous polypeptides were seen (fig. 3, lane 1). Instead, the cytoskeletal preparations of endothelial cells (fig. 3, lanes 2, 3) showed a major polypeptide with an apparent MW of 58 kD and some polypeptides of both lower (43 kD, corresponding to residual actin, [13, 141) and higher MW (e.g. 220 kD, corresponding to fibronectin [19]). The major 58 kD polypeptide, remaining in the cytoskeletal preparations of endothelial cells, comigrated with purified vimentin from human fibroblasts (fig, 3, lane 5) and could be identified as vimentin by the immunoblotting method using vimentin antibodies and the PAP-technique (fig. 3, lane 6). DISCUSSION In the present study we cultured human umbilic~ vein endothelial cells for several generations in the presence of FGF. Under these conditions endothelial cells were shown to undergo a disorganization of vimentin filaments which was independent of the organization of microtubules, and led into formation of juxtanuclear vimentin aggregates. Intermediate filaments appear to be closely associated both with cell nuclei [13, 20-221, with some cytoplasmic organelles [23, 241and also with the cell surface membrane [25, 261. Vimentin type of intermediate filaments are typically rearranged into perinuclear bundles in cells exposed to antimitotic drugs [S, 27-321 and this has been proposed as evidence for an interaction between microtubules and intermediate filaments [23, 25, 32-351. Also a direct coalignment between microtubules and inter-

Vimentin jilumrnts mediate filaments has recently been proposed using indirect immunofluorescen~e microscopy [36, 371. In our study endothelial cells from primary cultures and from early passages (2-7) showed a distribution of vimentin filaments reminiscent to that shown in earlier studies [ 1, 3-51. .The formation of spherical vimentin aggregates in late-passage cells, on the other hand, has not been observed earlier either in endothelial cells ‘or in other nontransformed cells. Such aggregates, consisting of both vimentin and keratin, have been recently described as a stable characteristic in a rat hepatoma cell line [38J and consisting only of keratin in a mouse epithelial cell line [39]. In the rat hepatoma cell line the juxtanuclear accumulation of intermediate tilaments was proposed to be a consequence of an association between vimentin filaments and the centrioles [38]. Such an association has been proposed also in dividing cells [40] and in virus-induced syncytia of cells [41]. In the present study we could; however, not demonstrate a direct association of centrioles with the vimentin aggregates in late passage cells, although centrioles could be found close to the vimentin aggregates. Moreover, our results show that vimentin filaments can undergo a complete disorganization without any effect on the cytoplasmic microtubular complex: ThiA supports the view that the organization of vimentin filaments is independent of microtubules as shown recently in cultured fibroblasts after disruption of microtubules by cold treatment [42] and by microinje?tion studies using monoclonal antibodies against intermediate filaments [43,#] In guinea pig endothelial cells colchicine has been reported to induce juxtanuclear cap-like aggregates of intermediate tilaments [4, 51, whereas in cultured human

in endothelial cdls

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endothelial cells, such a treatment has been shown to induce perinuclear whorls or coils of vimentin filaments [I]. In this study vimentin filaments in late-passage endothelial cells responded differently to treatment with demecolcine in comparison to ceils in primary culture. The latter cells showed typical coils of vimentin filaments, whereas in late-passage cells vimentin filaments aggregated into solitary masses. A reason for such a different response could be altered interaction of vimentin filaments with some other cellular component(s) responsible for normal segregation of intermediate filaments. This is suggested also by our results on spreading human endothelial cells: both cells in early and late passages showed typical juxtanuclear phase-dense aggregates of vimentin during the early spreading stage in line with results on porcine aortic endothelial cells [6] and on other cultured cells [22, 32, 341. In late passagecells, however, such aggregates apparently failed to be dispersed during cellular spreading. The aggregation of vimentin filaments appeared to be reversible by treatment of the cells with sodium butyrate. .Such results have also been described in a rat hepatoma cell line [38] in which sodium butyrate caused a dispersion of both vimentin and keratin aggregates. Other studies have additionally shown that treatment of malignant cells with sodium butyrate causes
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termediate filaments during cellular spreading and could be “repaired” by treatment of the cells with sodium butyrate. Late passage human endothelial cells could thus also provide a useful model in eIucidating the interactions of intermediate filaments with other cellular components. MS Riitta Vlistinen, MS Pipsa Kaipainen and MS Raili Taavela are kindly acknowledged for technical assistance. This study was supported by the Finnish Dental Society, Finnish Foundation of Cancer Research, The Sigrid Juselius Foundation, The Finnish Medical Research Council and the Research Fund of Finnish Life Insurance Companies.

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