Cytokeratin filament modulation in pulmonary microvessel endothelial cells by vasoactive agents and culture confluency

TISSUE AND CELL, 1991 23 (2) 141-150 0 1991 Longman Group UK Ltd.

J. S. ALEXANDER,

W. F. PATTON,

M. U. YOON and D. SHEPRO”

CYTOKERATIN FILAMENT MODULATION IN PULMONARY MICROVESSEL ENDOTHELIAL CELLS BY VASOACTIVE AGENTS AND CULTURE CONFLUENCY Keywords: Pulmonary ethothelium,

endothelium, cytokeratin,

vimentin, vasoactive agents

ABSTRACT. Recently, bovine pulmonary microvascular endothelial cells (PMV) were shown to contain cytokeratin 8 and 19 intermediate filaments (Patton et al., 1990). In this study, we examine the effect of culture contiguity and vasoactive agents on the content and assembly of cytokeratins in PMV. Immunofluorescent staining of PMV cultures show a progressive increase in cytokeratin filament assembly. In freshly plated PMV, keratin appears as hazy staining (<4 hr) and later organizes into keratin ‘plaques’ (4 days) associated with cell-cell contacts; post confluent (>7 days) PMV cultures contain fully assembled cytokeratin filaments which extend to the cell periphery and approach filaments in apposed cells. Vimentin filaments are also present in freshly plated PMV cultures but unlike cytokeratins, become less filamentous at confluency. This cell density-dependent modulation of cytokeratins is also demonstrated by densitometric analysis of autoradiographs of r5S-methionine labeled keratins in which PMV keratin content is elevated at high cell densities, while vimentin content remains constant. Desmoplakins I and II, components of desmosomes, could not be demonstrated in PMV by immunoblotting. PMV treated with permeability modulating agents (4 x lO_jM EGTA, 1 nM cytochalasin B. 1 pM bradykinin, 1 PM A23187, and 1 PM PMA) exhibit border retraction and altered keratin filament containing intermediate

staining. filaments

From these studies we conclude: 1) cytokeratin 8 and 19 are present in confluent PMV cultures with vimentin but

without desmosomes, 2) the state of assembly of PMV cytokeratin and vimentin filaments appears to be oppositely affected by culture contiguity, and 3) treatment of monolayers with vasoactive agents alters the state of assembly of cytokeratin filaments. We speculate that modualtion of cytokeratin assembly microvascular structure and function.

Introduction

Previously, we reported the presence of cytokeratin 8 and 19 containing intermediate filaments and vimentin intermediate filaments in cultured bovine pulmonary microvessel endothelial cells (PMV) (Patton et al., 1990). While most mammalian endothelial cells express only vimentin, cytokeratin expression in PMV is one of several documented examples of unusual intermediate Correspondence should be addressed to: Dr. J. S. Alexander, Box 1510, Station B, Vanderbilt University, Dept. of Biomedical Engineering, Nashville, TN 37235, USA. ’ Reprint requests to: David Shepro, Lab for Microvascular Research, 5 Cummington St., Boston, MA 02215. USA. Received 31 August Revised 31 October

1990 1990.

in PMV may be involved

in regulation

of pulmonary

filament expression patterns in vascular endothelium. Other reports show that cytokeratins are expressed in vivo in human synovial and sub-lingual mucosal endothelial cells (Jahn et al., 1987)) in bovine pulmonary microvascular endothelial cells in situ, in eel rete mirabile endothelial cells in situ (Mineau-Hanschke et al., 1990) and in essentially all endothelial cells in Xenopus (Jahn et al., 1987). Mammalian endothelial cells, thus far studied, which express simple epithelial cytokeratins also contain vimentin. Interestingly, this pattern of intermediate filament expression is consistent with that observed for many simple squamous epithelia (Franke et al., 1981). These atypical intermediate filament expression patterns in unusual endothelia may represent specialized adaptations of these vascular beds. 141

142

ALEXANDER

In epithelia, cytokeratin filaments associate with desmosomes, and together they regulate the organization of the macula adherens junction. This association contributes to tissue structural integrity, and may aid epithelial barrier maintenance (Bologna et al., 1986; Green et al., 1987; Franke et al., 1981). Jahn et al. (1987) report however, that desmosomes are absent in keratin positive (synovial and sub-lingual) human endothelia. Thus, the role of cytokeratins in PMV remains unclear. Since cytokeratins are modulated in PMV they may contribute to regulation of pulmonary microvascular function (Patton et al., 1990). In view of their potential importance, we have examined how keratins in PMV relate to endothelial barrier in two ways: 1) cell density/contact modulation of cytokeratin content and filament assembly in PMV and 2) modulation of cytokeratin filament assembly in PMV treatment by vasoactive agents.

Materials and Methods

ET AL.

were prepared after Chung-Welch et al. (1988) and characterized as described. PMV cultures were shown to be free of mesothelial contaminants based on their ability to form tube-like structures in three separate in vitro angiogenesis assays (Chung-Welch et al., 1989). Furthermore, it is observed that bovine PMV express cytokeratins 8 and 19 exclusively while pericardial mesothelial cells express cytokeratins 8, 18 and 19 (ChungWelch et al., 1989). AEC and PMV were maintained in medium consisting of DMEM, supplemented to 10% with FCS, and to 0.1% with Penicillin-Streptomycin-Amphotericin (Sigma). Cultures were subpassa ed weekly at 1: 4 split ratio (-2 X lo4 cells Bcm*). Cultures were re-fed twice a week with fresh medium. PMV and BAE cell cultures used in these experiments had been passaged between five (P-5) and eight times (P-8). Madin-Darby canine kidney cells (MDCK) were obtained as a gift from Dr. Donald Bottaro, NIH, Bethesda, MD and were maintained as described in DMEM supplemented to 10% with FCS and supplemented to 0.1% DMEM with PenicillinStreptomycin-Amphotericin.

Materials: Culture

media consisted of Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Gaithersburg, MD) supplemented to 0.1% with Penicillin-Streptomycin-Amphotericin supplement (Sigma) and to 10% with fetal calf serum (FCS, Gibco BRL, Gaithersburg, MD). Test agents (bradykinin, cytochalasin B (CB), EGTA and phorbol myristate acetate (PMA), and forskolin were purchased from Sigma Chemicals (St. Louis, MO). Calcium ionophore (A23187) was purchased from Calbiochem Corp. (San Francisco, CA). Stock solutions for CB, PMA, and forskolin were prepared as 1 mM solutions and diluted immediately before use, all other reagents were stored dessicated at -20°C prior to use. Cell culture: Bovine aortic endothelial cells (AEC) were harvested by ablation and 0.1% collagenase digestion by the method of Shepro et al. (1974). Cultures were confined to be ‘endothelial’ based on method of isolation, polygonal morphology, presence of factor VIII and uptake of acetylated low density lipoprotein (LDL). Pulmonary microvessel endothelial cell cultures (PMV)

ImmunoJEuorescence

microscopy: Confluent cultures of AEC or PMV were trypsinized and suspended at 25% density in cell culture media and seeded onto 1.2 cm diameter glass coverslips in 24 x 1 ml limbro wells (Falcon). Cultures were grown to the density state described for the experimental protocol, and treated as described. After treatment, coverslips were fixed in -20°C acetone/dry ice for 3 min. Extracted coverslips were incubated with 50~1 of either monoclonal anti-cytokeratin 8 (clone 8.12, Sigma Corp., St. Louis, MO), monoclonal anti-cytokeratin 19 (clone 4.62, Sigma Corp.) or monoclonal antivimentin (clone VIM-13.2, Sigma Corp.), diluted 1: 50 in PBS plus 1% milk powder for 1 hr. Coverslips were washed 5 times (5 min each wash) in PBS. Coverslips were stained with 50 ~1 of rhodamine labelled goat anti-mouse antibody (30 pg/ml) for 1 hr. Coverslips were washed 5~ in PBS, postfixed in 3.7% formaldehyde for 5 min and washed in PBS. Coverslips were mounted in 5 ~1 of 1: 1 glycerol/PBS (with % 0.1% pphenylenediamine) and illuminated for fluorescence using an IM-35 microscope

143

MODULATION OF PULMONARY CYTOKERATINS

(Carl Zeiss, FDR). Photographs were all recorded at 1000x onto T-MAX 3200ASA film (Kodak Co., Rochester, NY) and processed with Unit01 developer (JOB0 Corp., FDR). Photographs were printed on Kodabrome F-M paper. Electrophoresis, immunoblotting, and image analysis: Polypeptides from pre-confluent

through post-confluent cultures of AEC and PMV were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% T, 2.6% C matrix (Laemmli, 1971). For 3SS-methionine labeled samples, 50,000 dpm of trichloroacetic acid precipitable counts were loaded per lane while for immunoblot analysis 100 ,ug of protein was loaded per lane. Polypeptides were electrophoresed at 15 mA constant current and for 35S-methionine labeling studies, subsequently incubated for 30min in two changes of dimethylsulfoxide, followed by a 3 hr incubation in 20% 2,5-diphenyloxazole (PPO) in dimethylsulfoxide, water for 1 hr and then in 2.5 x glycerol for 2 hr prior to air drying between dialysis sheets (Dunbar, 1987). Radiolabeled gels were exposed to Kodak X-OMAT film following manufacGels were analyzed turer’s instructions. using a microcomputer-based videodensitometer as previously described (Patton et al., 1989). For immunoglocial detection of the cytokeratins and the desmoplakins I and II, gels were transferred to nitrocellulose by the method of Towbin et al. (1979) as modified by Dunbar (1987). Protein transfer was confirmed by visualizing the proteins with Ponceau S prior to incubation with monoclonal antibodies. The nitrocellulose membrane was then incubated in 5% dry milk for 2 hr, and in mouse anticytokeratin antibody diluted 1: 150 (Sigma, clone 4.62), or in antiDesmoplakin I-II antibody (clone DPl and 2-2.215, Boehringer-Mannheim Biochemicals, Indianapolis, IN) diluted 1:150 for 12 hr. After washing, blots were incubated in 1: 500 diluted alkaline phosphatase conjugated goat anti-mouse antibody containing 0.1% milk powder for 2 hr. The alkaline phosphatase was then visualized by incubating for 15 min in a nitroblue tetrazolium chloride/5-bromo-4-chloro - 3 - indolyphosphate p-toluidine chromogen system.

Results Intermediate filament assembly as a function of cell confluency and length of time in culture:

Cytokeratin and vimentin distribution in PMV was greatly influenced by the state of confluency and age of PMV cultures. In freshly plated cells allowed to attach and spread for 4 hr on the culture dish, vimentin was distributed as filaments which extend radially from the cytocentrum to the cell limit (Fig. 1A). Cytokeratins 8 or 19 localized in PMV after 4 hr are observed as diffuse, hazy staining throughout the cell (Fig. 1B) suggesting filament disassembly. At 50% confluency, (4 days in culture), most cytokeratin 8 and 19 staining in PMV was observed at the intercellular junction, similar to ‘keratin plaques’ (Fig. 1D) described for epithelial cell types (Bologna et al., 1986). Keratin plaques appear dependent on cell-cell contact, free cell margins not in contact with cells do not show plaque staining (see arrow). After 10 days in culture, post-confluent PMV cultures exhibit distinct cytokeratin 8 and 19 bearing filaments throughout the cytoplasm (Fig. 1F). Unlike cytokeratins, vimentin filament assembly in PMV is not persistent in culture, and post-confluent cultures of PMV (10 days) show only diffuse cytoplasmic staining and lack discrete vimentin filaments (Fig. 1C). Bovine aortic endothelial cells (AEC) express only vimentin intermediate filaments, with no staining for cytokeratin (Patton et al., 1990). Vimentin filaments in AEC are present at pre-confluency through postconfluency form a complex network throughout the cell (Fig. 1E). Desmoplakins I and II were found to be present in Madin-Darby canine kidney epithelial cell controls, but could not be demonstrated in PMV by immunoblotting techniques (data not shown). Intermediate filament content in PMV as a function of cell density: The relationship

between cell seeding density and keratin expression was also examined by densitometric analysis of cytokeratin 8 and 19 immunoblots. Keratin expression in PMV relative to total protein was found to he markedly influenced by cell density (Fig. 2). Cells seeded at 2.5 x 105 cells/cm* (confluent seeding) show an approximate 4-fold increase in cytokeratins 8 and 19 over cells seeded at 1.25 x 104cells/cm2 (sparse seed-

MODULATION

OF PULMONARY

145

CYTOKERATINS

(1 PM, 15min) (Fig. 4f). This treatment induces cell retraction and arborization by microfilament disintegration (Shasby et al., 1983); cytokeratin filament staining shows a similar concentration of cytokeratins at the cytocentrum. Filaments were observed in both cases which extend to points of cell-cell contact, and frequently show close approach between adjacent cytokeratin bundles (Fig. 3).

Denslty Dependent Modulation of Intermediate Filament Proteins

Effects of vasoactive mediators on PMV intermediate filaments: Control cytokeratin

0.0

! 0

I

I

1x102

2x106

1

2x102

Cells/cm2 Fig. 2. Effect of culture seeding density on PMV expression of cytokeratins and vimentin. The amounts of the intermediate filament proteins are expressed relative to actin. Results of densitometric scans of cytokeratins 19 and 8 were averaged and plotted as Average cytokeratins. Increasing seeding density leads to increased expression of cytokeratin filaments in PMV as measured by densitometric analysis. Vimentin expression remained essentially unaltered by cell density.

ing). As the cultures become progressively more cell-dense, the amount of keratin in their cytoplasms steadily increases. Vimentin content in PMV is not altered significantly through post-confluency, despite the morphological changes observed by fluorescence microscopy. Association of cytokeratin filaments with the PMV endothelial junction: Figure 3 shows

the distribution of cytokeratin 8 in a control monolayer. Filaments are visible which extend from the central keratin network to intercellular contacts, where cell-cell contacts contain assembled keratin filaments. A similar appearance is observed in PMV monolayers treated with cytochalasin B

distribution: Monoclonal antibodies to cytokeratin 8 and 19 co-localize to the same filament networks in cultured PMV and show almost identical staining patterns in PMV. In examining the effects of vasoactive agents, we focused on the distribution of cytokeratin 8 filaments in cultured PMV. However preparations stained for cytokeratin 19 show similar filament distributions under baseline conditions and following various treatments (Fig. 4A). A23187 (calcium ionophore): PMV treated for 30 min with 1 PM caclium ionophore (A23187) showed retraction of cytokeratin filaments with increased staining intensity in the perinuclear region. During retraction, cytokeratin filaments lose some filamentous appearance and show diffuse staining (4B). Bradykinin: PMV treated for 30 min with 1 PM bradykinin, showed central retraction of cytokeratins with an apparent loss of filament assembly. Numerous cytokeratin filaments can be seen which remain extended to intercellular contacts (4C). EGTA: PMV treated for 2 hr with the calcium chelator EGTA (4mM) in DMEM, showed extensive cell border retraction and replacement of filaments by hazy staining for cytokeratins 8 and 19 (4D) when compared to cells maintained in normal calcium supplemented media phorbol myristate acetate (PMA): PMV treated for 30 min with 0.1 PM

Fig. 1. Immunofluorescent staining of intermediate filaments in cultured bovine pulmonary microvessel (PMV) post seeding. A) Vimentin filaments in PMV 4 hr post seeding. B) Cytokeratin staining in PMV 4 hr post-seeding. C) Vimentin staining in PMV 3 days post confluency (7 days post seeding). D) Early confluency (3 days post-seeding) ‘keratoplaques’ (cytokeratin 19 staining). E) Confluent staining pattern of vimentin filaments in cultured bovine aortic endothelium. F) Confluent staining pattern of cytokeratin (19) in PMV (7 days post-seeding). X1000.

146

ALEXANDER

ET AL.

Fig. 3. Association of cytokeratin filaments with the endothelial junction. PMV culture shows distinct cytokeratin bundles extending into intercellular contacts. Note junctional space between filaments. x 1ooO.

phorbol my&ate acetate (PMA) (4e) showed monolayer retraction and a marked loss of cytokeratin filaments when stained for either cytokeratin protein. Cytochalasin B (CB): Monolayers treated with CB at 1 PM showed retraction of the cytokeratin filament system (4f). CB treated cells show a retracted appearance but still often maintain intercellular contact regions where cytokeratin bundles approach closely but do not contact.

Fig. 4. Immunofluorescent

staining

Discussion

Our findings suggest that cytokeratin intermediate filaments in PMV cytokeratins are not static structural elements, but dynamic cytoskeletal constituents whose synthesis and assembly is altered by culture density and vasoactive agents. Unlike microfilament and microtubule based structural systems, intermediate filaments (e.g. cytokeratins) are traditionally regarded as ‘true’ cytoskeletal

of cytokeratin

containing

fibers in cultured

bovine

PMV

treated with vasoactive agents. (A) control treated (DMEM alone) treated PMV, 15 min. (B) 1 pM A23187 treated PMV, 15 min. (C) 1 pM bradykinin treated PMV, 15 min. (D) 4 x 10m3M EGTA treated PMV, 2 hr. (E) 1 pM phorbol myristate acetate (PMA) treated PMV, 15 min. (F) 1 pM cytochalasin B treated PMV, 15 min. x1000.

ALEXANDER

elements, i.e. structural, non-motile elements whose motion and arrangement passively parallels other cell motile elements (Lazarides, 1980). However, recent reports show that all intermediate filament classes may be structurally modified by phosphorylation and during cell division (Celis et al., 1983; Inagaki et al., 1987; Tokui et al., 1990). Thus, the state of assembly of intermediate filaments could serve as an important means of regulating cell shape and endothelial barrier function in PMV. Following trypsinization and re-plating, we followed the recovery of the mature appearance of cytokeratins in PMV. Pre-confluent PMV cells contain small amounts of cytoplasmic keratin, which is not assembled as filaments. Formation of cell-cell contacts appears to induce PMV cytokeratin synthesis and assembly. In PMV cultures near confluency cytokeratin staining is observed as peripheral keratin ‘plaques’ (Bologna et al., 1986) where cell contacts are formed. Free cell margins in these cultures fail to show cytokeratin plaque staining. Peripheral keratin later matures to discrete cytokeratin bundles in post-confluent PMV cultures which extend throughout the cytoplasm and retain edge contacts. Cell-cell contact mediated keratin organization suggests that cytokeratin filaments interact with and arrange PMV junctions as they do in epithelial junctions (Green et al., 1987; Bologna et al., 1986). Cytokeratin content in PMV is also elevated with increasing culture density. Confluent cultures display a 4-fold increase in cytokeratins over subconfluent cultures and the induction of cytokeratins by cell-cell contact seems to indicate a specific role of cytokeratins in the functioning of confluent endothelial monolayers. Inter-endothelial junctions link cells within the confluent monolayer to treat an exchange barrier to the compartments separated by the monolayer. Prior to confluency, endothelial junctions do not exist, and thus monolayers cannot maintain a permeability barrier. Therefore, PMV junctional apposition and cytokeratin regulation in confluent PMV appear to be related. Such interactions could provide an additional mechanism for regulating pulmonary microvascular barrier function. In addition to chronic structural regulation by culture density, the effects of acutely act-

ET AL.

ing mediators on cytokeratin assembly was also examined in PMV. Monolayers treated with the calcium chelator EGTA (4 mM) for 2 hr exhibit retraction of cell borders and a dramatic loss of cytokeratin filament staining. Thus, within PMV, calcium dependent intercellular contacts may alter keratin assembly as they do within desmosomes et al., 1986). The identity of (Bologna specific junctional protein(s) associated with cytokeratins in PMV have not yet been determined, however our findings exclude desmosomal proteins (e.g. desmoplakin I, II) as possible PMV junction constituents. Cytokeratin expression without desmosomes (Jahn et al., 1987) occurring in endothelia as PMV suggest that cytokeratins may associate with, and organize non-macula adherens type contacts, e.g. focal adhesions (Bershadsky et al., 1985), or 6 nm zonula adherens (Franke et al., 1988). Endothelial cell activation by inflammatory mediators is thought to involve calcium mobilization and protein kinase C activation (Ryan et al., 1988; Haselton et al., 1990) and such activation is reported to affect cell shape and cytoskeietal assembly in several system (Welles et al., 1985; Meigs and Wang, 1986; Inagaki et al., 1987). We observe that PMV cultures treated with inflammatory agents which mobilize cell calcium (bradykinin, A23187), or which activate protein kinase C (PMA) exhibit rapid retraction of cell-cell apposition and retraction and dissolution of cytokeratin filaments. Thus, changes in cytokeratin filament organization and assembly promoted by these mediators could provide a means for regulatory pulmonary microvascular structure and permeability. Whether changes in cell-cell apposition causes changes in filament assembly is not clear, yet EGTA results suggest that loss of cell contact mediates filament assembly in PMV (Bologna and Dulbecco, 1986). Cytochalasin B is a potent microfilament toxin which promotes vascular permeability in vitro by promoting endothelial actin filament dissolution (Shasby et al., 1983). Since cytochalasin B promotes central collapse of PMV keratins without apparent alteration of cytokeratin filament assembly, it appears that PMV keratins associate with other filament systems e.g. microfilaments yet maintain independent structural regulation (Knapp and Bunn, 1987).

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OF PULMONARY

149

CYTOKERATINS

It is observed that 4 hr after replating, PMV exhibit vimentin as assembled filaments, unlike cytokeratins staining which is diffuse. In large vessel endothelial cells (AEC) a constant level of vimentin is maintained independent of culture density. Vimentin filament assembly state in AEC is also density independent and filaments are apparent at all phases of culture. Conversely, PMV vimentin filaments become less distinct at confluency, although total vimentin content in PMV remains unchanged. Thus, vimentin might participate in coordination of sub-confluent cell activities as cell division and motility, rather than in barrier formation in confluent PMV. AEC and PMV vimentin behavior differs in large and microvessel endothelia, and suggests that independent functions exist for these intermediate filament components. Large vessel endothelial cells e.g. AEC exist in a high shear, low tension environment, whereas the pulmonary microvessel experiences low shear with constant cylical tension due to ventilation (Patton et al., 1990). The structural arrangement of keratin

filaments in epithelia disperses tension in the tissue; it is also possible that keratins within PMV fulfil a similar function. Cytokeratins are major components of intermediate filaments in epithelia (Franke et al., 1981; Moll et al., 1982) also present in some types of specialized endothelia (Jahn et al., 1987; Patton et al., 1990). Although the function of cytokeratins in pulmonary microvessel endothelial cells are not known, evidence suggests that this structural system organizes cell-cell and cell-substrate associations to maintain and enhance pulmonary microvascular integrity. Continued research on this cell structural component may contribute to improve understanding of lung vascular physiology in vivo. Acknowledgements

The authors acknowledge Dr. F. R. Haselton for superior assistance in preparation of this manuscript and Dr. Nancy Chung-Welch for supplying bovine PMV endothelial cells for these experiments. This work was supported in part by NHLB grants HL16714, HL43875.

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