Differentiation (1991) 46:117-133

Differentiation

Ontogeny, Neoplasia and Differentiatinn Therapg

0 Springer-Verlag 1991

Microvessel endothelial cell transdifferentiation: phenotypic characterization Bruce H. Lipton”, Klaus C. Bensch, and Marvin A. Karasek Departments of Dermatology and Pathology, Stanford University, School of Medicine, Stanford, CA 94305, USA Accepted in revised form December 31, 1990

Abstract. Human dermal microvessel endothelial cells (MEC) have two basic functions : maintenance of tissue homeostasis and facilitation of inflammatory reponses. The former requires that the endothelium expresses traits of an epithelium, while inflammatory reactions are associated with intimal disruption. Acute inflammation transiently alters endothelium, whereas chronic inflammation may result in vessel reorganization and MEC mesenchymalization. Foreskin MEC in vitro undergo a similar epithelial-mesenchymal modulation. In the presence of CAMP, cultivated dermal MEC exhibit the structural and functional characteristics of an epithelium. MEC grown in CAMP-deficient medium initially have a “transitional” configuration and are subsequently transformed into mesenchymal cells. If CAMP is replaced by histamine, MEC maintain a stable intermediate transitional configuration. Transitional MEC refed CAMP-supplemented medium revert to an epithelial phenotype, whereas parallel cultures fed CAMP-deficient medium are transformed into mesenchymal cells. Phenotypic modulation can be induced without cell division and thus provides a unique example of direct transdifferentiation. Our data furthermore suggest that this transdifferentiation results in the acquisition of properties usually attributed to cells of the reticuloendothelial system.

Introduction Endothelial cells of the microvasculature participate in a wide variety of regulatory, synthetic and secretory activities [21, 48, 50, 531. Despite their seeming diversity, all of these activities can be subdivided into supporting either of two fundamental vessel functions: homeostasis or inflammatory reactions. Of these, homeostasis is dependent upon microvessel endothelial cells (MEC) expressing an epithelial phenotype [49, 531. In this cellular

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configuration a vessel is endowed with membrane properties necessary for regulating the exchange of molecular constituents between the blood and the surrounding tissue [ 10, 481. Major tissue and cellular perturbations compromising homeostasis usually result in an inflammatory or defense response, which may vary with the causative agent or type of injury [5, 591. In switching from a homeostatic to an inflammatory-reactive role, MEC lose their epithelial character [33, 441. These phenotypic changes are associated with a remarkable cytological reorganization, accompanied by a loss of junctional contiguity and disruption of the intimal lining [21, 31, 39, 481. In this setting, vascular permeability alterations result in fluid exudation (edema), margination (binding) and emigration of differing types of inflammatory cells, clotting phenomena and ultimately even tissue necrosis [31, 46, 501. While acute responses are transient and reversible, chronic inflammatory processes may induce permanent MEC phenotypic changes resembling epithelial-mesenchymal transformation [3, 38, 441. While such transformations are normally associated with embryogenesis [ 17, 18, 191, the observed inflammation-induced modulation may more appropriately reflect an example of epithelialmesenchymal transdifferentiation [4]. The term transdifferentiation refers to an alteration in the phenotype of a cell that had previously acquired a stable cytodifferentiative character [4, 43, 511. Though formerly considered a rare phenomenon, transdifferentiation has been documented with increasing frequency in recent years (reviewed in [4]). Rather than invoking irreversible genomic alterations as a means of stabilizing differentiative expression, transdifferentiation studies suggest that the extracellular environment, the cytoplasm and components of the nucleus interact to confer stable gene expression [51]. In transdifferentiation these interactions are interrupted and the affected cells are induced to alter their phenotypic expression. Investigations of model systems expressing either epithelial-mesenchymal transformations or transdif-

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ferentiations have led Hay [17, 18, 191 to postulate the existence of master-genes that are activated during epithelial-mesenchymal transformations, and which in turn switch on tissue-specific effector gene sets. This report focuses on the phenotypic characterization and regulation of an epithelial-mesenchymal transdifferentiation model which confirms and extends Hay's master-gene hypothesis. Our studies demonstrate that dermal MEC in cell culture can express both epithelial and mesenchymal configurations, as well as an intermediate " transitional-cell" form. While details of dermal microvascular endothelial cell (MEC) ultrastructure have been described before [Z, 6, 571, this report expands those studies to focus on the mechanisms controlling phenotypic expression. MEC phenotypic modulations are differentially responsive to cAMP concentration and activity of Ca 2+ second messenger systems. The MEC cell system provides a novel in vitro model of direct transdifferentiation, since the described phenotypic modulation can occur in the absence of cell division. Furthermore, the results presented indicate that epithelial-mesenchymal modulations induced by inflammatory stimuli may lead to the expression of functions currently attributed to cells of the mononuclear phagocytic system, formerly the reticulo-endothelial system [I].

Fluorescence assays. For cytoskeletal studies, cell cultures were fixed with 2.5% formaldehyde made freshly from paraformaldehyde in PBS and subsequently permeabilized with 0.5% Triton X-100 in PBS. Cells were doubly stained with mouse monoclonal antibodies to vimentin (Clone-VY, Boehringer Mannheim) and actin-specific rhodamine-phalloidin (Molecular Probes) according to manufacturer's specifications. Von Willebrand (factor VIII) antigen, used to assess the cytodifferentiative expression of cultivated MEC, was assayed with monoclonal mouse antibodies (Dakopatts). Primary antibodies were detected with complementary fluorescein isothiocyanate (F1TC)-conjugated antiglobulins (Dakopatts) in glycerol-mounted specimens, and examined in a fluorescence-equipped Nikon Micropbot F X microscope. Collagen gel induction. The luminal surface of control and experimental M E C cultures were covered with a layer (approximately 1 mm thick) of type I collagen (Vitrogen) and 1OX minimal essential medium (MEM) in a 10:l ratio [14, 251. Following incubation a t 37" C for 30 min, the polymerized gels were covered with medium. Cells were observed using phase microscopy, and at selected timcs cultures were fixed for either light or electron microscopy.

Results Epithelial characteristics of MEC cultivated i~ C A M P supplemented medium

MEC adhere to and flatten upon gelatin-coated substrates within 4 h of plating. When grown in CAMP-

Methods Standard M E C cultures. Dermal microvascular endothelial cells were isolated from neonatal human foreskin and grown on collagen-coated substrates in a modified Iscove's growth media supplemented with 8% newborn calf serum, 2% prepartum maternal M isobutyl methyl serum, 5 x M dibutyryl CAMP, 3.3 x xanthine (IMX), 1 x M hypoxanthine and 1 . 5 ~ M thymidine [22]. MEC maintained in this medium express structural traits, including factor VIII antigen, Weibel-Palade bodies, tight junctions, and pinocytotic vesicles, closely resembling endothelial cells in vivo [2, 221. Cell growth and morphology were monitored with phase microscopy. Third through fifth generation MEC were routinely used in these studies. Moduluted M E C cultures. Transitional and spindle-shaped MEC were obtained by replacing standard growth media with medium deficient in cAMP and IMX [57]. Proliferating and confluent MEC cultures present identical phenotypic responses. Histamine- und cytokine-treuted M E C cultures. Proliferating or confluent endotbelial cultures (third through fifth passage) were refed CAMP-free medium with and without M histamine (Sigma). In some studies, MEC exposed to CAMP-free medium containing 2 U/ml Interleukin-I (Boehringer Mannheim) and/or 500 U/ml human recombinant ?-interferon (Biogen) were used to assess the influence of cytokines on transitional cell expression. Irradiated cell cultures. To determine whether cell division is required for modulation of MEC expression, confluent monolayers of MEC in 16-mm wells were exposed to 24 Gy from a cesium source. Control and experimental cultures were fed either 2 h before or immediately after exposure. Proliferation in the treated and control cultures was determined using a bromodeoxyuridine (BrdU)/anti-BrdU antibody assay (Cell Proliferation Kit RPN 0.20, Amersham). Electron microscopy. Cultivated MEC were fixed with 3% glutaraldehyde [2], postfixed in buffered 1% OsO,, stained with uranyl and lead salts and were examined in an Elmiskop 101 electron microscope.

Fig. 1 A-D. Morphology of cultured foreskin-derived dermal microvessel endothelial cells (MEC). A MEC in actively growing cultures proliferate and form small plaque-like colonies. Mature cells exhibit a flattened polygonal morphology with a dense central zone containing the nucleus and most of the membranous organelles. The attenuated peripheral margins are characterized by low cytoplasmic density. Newly derived less-differentiated daughter cells accumulate at the periphery of the epithelial plaques. The immature, bipolar cells have a taller profile and optically appear to have a denser cytoplasm. The distinctive bright margins around bipolar cells reflect a lack of epithelial junctional specialization. Melanocytes (arrowheads) comprise approximately 5% of the population and are distinguished by their narrow spindle-shaped or flattened stellate configuration. B Confluent MEC culture at stationary phase of growth. MEC possess typical flattened squamous cell morphology with thicker central zone containing most organelles. All epithelial cells are positive for factor VIII (von Willebrand factor) antigen and possess Weibel-Palade bodies. Zonular occluding junctions, observed in electron microscopy, are responsible for apparent cytoplasmic continuity at peripheral cell margins. These cells exhibit contact inhibition of growth and maintain a monolayer with no cellular overlap; arrowhead, melanocyte. C MEC in confluent cultures exposed to CAMP-free medium for a t least 6 h or more, or exposed to M histamine for less than 5 min after I h preincubation in CAMP-free medium, exhibit distinct morphological alterations. In this micrograph, MEC have been deprived of CAMP for 8 h. Junctional dissolution and cell contraction pro'duce gaps in the epithelioid sheet. Individual cells assume the characteristic broad, flattened bipolar configuration of transitional cells (arrows). D M E C convert into true spindle-shaped cells in approximately 1 week of inducing the bipolar transitional cells in CAMPfree medium. Spindle-shaped cells express mesenchymal characteristics, which include multilayering of cells, synthesis of collagens I and IT1 and expression of immune-related surface antigens. Broad lamellipodia (arrow) indicate anterior, or leading, end of migratory spindle-shaped cells. Once MEC acquire this morphology, they no longer express endothelial properties even upon return to normal, CAMP-supplemented growth conditions

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Fig. 2A, B. Fine-structural features of epithelioid MEC in vitro. A Transverse section through epithelioid MEC, revealing ultrastructural characteristics of thicker, central nucleus- and organellecontaining region. Characteristic organellar stratification includes endothelial-specific Weibel-Palade bodies (urruws) beneath apical plasmalemma, supranuclear Golgi bodies, nucleus and subnuclear rough endoplasmic reticulum. Organelles are enmeshed in a reticular intermediate filament (vimentin) matrix. Dotted profiles (arruwheads) indicate cross-sections of intermediate filament bundles. B

Cross-section through MEC at region where thickened central organellar zone undergoes transition into flattened peripheral cell margin. Dense, coarse bundles of intermediate (vimentin) filaments (arrows) characteristically demarcate these two zones (see Figs. 3 B and 4A). Peripheral margins contain pinocytotic vesicles and occasional mitochondria and Weibel-Palade bodics in woven matrix of intermediate and microfilaments. Epithelioid MEC secrete extracellular, basal lamina-like matrix on the culture substrate

supplemented medium, MEC replicate, and reflatten to form circular epithelioid colonies. A zone of bipolar cells characteristically encompasses the enlarging epithelioid plaques (Fig. IA). Upon reaching confluency the cultures enter a stationary phase of growth. In such cultures the majority of MEC exhibit a characteristic simple squamous epithelial morphology (Fig. 1 B) and remain monolayered despite increased cell densities in proliferating cultures. Studies of MEC fine structure reveal that the nucleus and most of the organelles (mitochondria, endoplasmic reticulum, Golgi apparatus and lysosomes) are clustered in the thicker central portion of the squamous cells (Fig. 2). The organelles are differentially stratified and enmeshed within a matrix of interlaced 10-nm intermediate filament bundles (Figs. 2A, 3C). Endothelially specific Weibel-Palade bodies are dispersed in the subplasmalemmal matrix beneath the cell’s apical surface. The

Golgi bodies lie above the nucleus, while most of the endoplasmic reticulum is found in the basal portion of the cell (Fig. 2). The extensive attenuated peripheral margins of the epithelioid MEC primarily contain pinocytotic vesicles, mitochondria and Weibel-Palade bodies within a matrix of micro- (5- to 7-nm) and intermediate (10-nm) filaments (Fig. 2B). Neighboring cells adhere to each other at their lateral margins through zonular junction attachment complexes (Fig. 3A). An electron-dense matrix of 5- to 7-nm filaments is intimately associated with the cytoplasmic face of the junctional plasmalemma. This dense matrix encircles the entire perimeter of epithelial MEC and is comprised of actin (as demonstrated below). Fluorescence microscopy of intact MEC monolayers reveals that epithelial MEC possess an elaborate and characteristic cytoskeletal matrix. Vimentin forms a radially concentric matrix with two distinct zones

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Fig. 3. Fine structure of micro- (5- to 7-nm) and intermediate (10-nm) filaments in epithelioid MEC. A Junctional margin between two epithelioid endothelial cells; Plasmalemmal-associated microfilaments (actin, see Fig. 4A) form encircling dense peripheral bundle at sites of zonular junctional specializations. B Fluorescence micrograph of FITC-conjugated anti-vimentin-labelled MEC. vimentin in epithelioid MEC is organized into two zones: (1) coarse reticular vimentin matrix radiating from nuclear envelope enmeshes

organelles in central region; (2) a concentric zone of finer vimentin bundles subtends attenuated peripheral margins. An area of the cell similar to that at the tip of arrow is illustrated in C. C Region similar to that indicated by arrow in B. Interwoven bundles of 10-nm vimentin filaments form reticular matrix enveloping mitochondria, endoplasmic reticulum, lysosomes and Weibel-Palade bodies

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(Figs. 3B, 4A, C). Centrally, vimentin forms a thick equatorial belt close to the nuclear envelope. Coarse vimentin bundles radiating from this nuclear ring form a flattened spherical reticular zone surrounding the nucleus (Figs. 3B and 4A). Most of the cellular organelles are enmeshed within this vimentin reticulum (Fig. 3 C). Coarse vimentin filament bundles encircle the reticular portion of the matrix and form an outer concentric ring, which separates the matrix from the flat, broad cell periphery (Figs. 2B, 3B, 4A, C ) . This attenuated cell periphery possesses an irregular feltwork of thinner vimentin bundles (Figs. 3B, 4). Rhodamine-phalloidin reveals a characteristic organization of filamentous actin, principally restricted to dense peripheral bundles (DPB, see [60], which clearly delineate the cellular margins (Fig. 4A). Redundant DPBs reflect multiple rows of zonular intercellular junc-

Fig. 4. A-D Fluorescence micrographs of epithelioid, transitional and mesenchymal MEC doubly stained with FITC-conjugated anti-vimentin and actin-specific rhodamine-phalloidin. A Epithelioid MEC possess a characteristic actin and vimentin cytoskeleton. Actin, associated with junctional specializations, comprises a belt (DPB) which encircles the perimeter of the squamous MEC. Duplications of actin bundles reflect sites of membrane overlap and cellular interdigitations. Vimentin is deployed in two concentric zones surrounding the nucleus. Radiating from the nuclear envelope, thick bundles of vimentin create a circular, reticular network enmeshing most of the membranous organelles (see Fig. 3B, C). Finer vimentin bundles in the cell periphery form an irregular matrix, which extends to the cell margins. Note the presence of bipolar transitional cells along the edge of the epithelial plaque. B Within 10 min of exposure to histamine, MEC in CAMP-free medium express significant cytoskeletal alterations. Portions of the actin-associated DPB disappear as newly assembled actin stress fibers appear beneath the apical plasmalemma (see Fig. 5B). Dissolution and reorganization of the vimentin matrix (arrowheads) are associated with morphological alterations leading to a bipolar configuration. Junctional dissolution and cell contraction produce intercellular gaps (urrows).C After 24 h in CAMP-free medium, formerly epithelioid MEC express transitional phenotypes. A late-responding epithelioid endothelial cell (center-right)still exhibits a central reticular zone although its peripheral vimentin matrix has detached and retracted from the cell margin. Portions of the actin peripheral bundle are still evident along this margin (arrowheads). A typical migrating bipolar transitional cell (centev-left, urrow) possesses a wide lamellipodium and associated actin stress fibers at its anterior end. D MEC exhibit a mesenchymal spindle-shaped morphology after 5 days in CAMP-deficient medium. The actin and vimentin are organized as parallel linear arrays that traverse the long axes of the cells. E, F Factor VIII (von Willehrand) antigen distribution in epithelioid and transitional MEC. E In CAMP-containing medium, squamous epithelioid MEC express positive staining for factor VIII antibodies. Factor VIII antigen is present in both a diffuse cytoplasmic form and as discrete rod-like granules, whose distribution resembles that of Weible-Palade granules observed in fine structural studies. F Transitional cells in MEC cultures exposed to CAMP-free medium for 24 h. Transitional cells express a similar distribution, but reduced intensity for cytoplasmic factor VIII staining compared to epithelioid cells. However unlike control cultures, transitional cultures reveal the presence of an extracellular reticular matrix of factor VIII antigen, which envelops the cells. As MEC acquire a spindle-shaped mesenchymal morphology, cytoplasmic factor VIII staining is lost and staining of the extracellular matrix is concomitantly increased

tions brought about by overlapping and frequently interdigitated cellular margins. Clusters of short actin stress fibers are occasionally encountered in the center of larger, very flat epithelial MEC. Electron microscopy reveals that these stress fibers are anchored to the cytoplasmic face of the basal plasmalemma (not shown). Epithelial MEC are stained by Factor VIII antibodies (Fig. 4E). The granularity and frequency of the fluorescent deposits mirror the appearance and distribution of Weibel-Palade bodies as observed in the electron microscope (Fig. 2A). Mesenchymal characteristics of cA MP-deficient medium

MEC cultivated in

MEC expression is dramatically altered in cultures fed medium without CAMP. Approximately 5 h after CAMP withdrawal, sites of junctional dissolution lead to formation of intercellular gaps (Fig. l C). During the next 12 h cell retraction and enlarging gaps disrupt the epithelial monolayer. Individual MEC exhibit various degrees of contraction. While epithelioid cells have a greater tendency to completely round up and detach from the plate, bipolar MEC bordering the epithelioid plaques (see Fig. 1A) are more refractive to CAMP withdrawal. The resulting flattened bipolar MEC in CAMP-deficient cultures express the phenotypic characteristics of “ transitional cells” described below. If refed CAMP-supplemented medium, these bipolar cells proliferate and subsequently reestablish confluent epithelial colonies. However, if the cells are maintained in CAMP-free medium for 3 or more days, the transitional cells begin to convert into colonies of multilayered, rapidly proliferating spindle-shaped cells (Fig. 1D). These sharply demarcated, narrow cells are readily distinguishable from their broader and more-flattened bipolar MEC precursors. Spindle-shaped MEC are migratory and frequently possess from one to three pseudopodia1 structures with ruffled cell membranes at their leading anterior poles, The overlapping and multilayering of mesenchyme-like MEC is indicative of the loss of the contact inhibition properties (Fig. 1 D). Cultures containing spindle-shaped MEC are also metabolically different from epithelial MEC cultures. Within 3 days of refeeding, mesenchyme-like spindled cells acidify (pH 6.8) their culture medium while epithelioid and transitional cells maintain a near-neutral (pH 7.6) growth environment. Mesenchymal spindle-shaped cells do not exhibit zonular junctions and are devoid of any membrane specializations at sites of cell-cell contact (Fig. 5A). Endothelially specific Weibel-Palade bodies are strikingly absent, while the general incidence of all other organelles remains similar to that in epithelioid MEC. In these cells, the organelles and cytoskeleton have a characteristic distribution, which supports and conforms to the cell’s bipolar configuration. Cytoplasmic organelles in mesenchyme-like MEC are ordered in an anterior-posterior axis, rather than in the apical-basal stratification characteristic of epithelioid MEC. Ruffled

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membranes and lamellapodia identify the anterior end of the cell. In addition, the centrosomes of mesenchymal MEC are localized adjacent to the anterior-oriented side of the nucleus (not shown). Thin (5- to 7-nm) filaments form numerous stress fibers subtending the apical plasmalemma (Fig. 5B, D). Linear bundles of intermediate (10-nm) filaments, aligned with the thin-filament stress fibers, traverse the longitudinal axes of the bipolar cells (Fig. 5B). Fluorescence microscopy of tagged actin and vimentin filaments supports the fine-structural observations. In the mesenchyme-like spindle cells, both filament types are characteristically deployed in parallel arrays traversing the anterior-posterior axis of the cell (Figs. 4D, SC). Anti-factor-VIII staining reveals that spindle-shaped cells are loosely surrounded by factor-VIII-positive extracellular deposits (see Fig. 4F). The observation that mesenchyme-like MEC do not contain cytoplasmic factor VIII antigen correlates with the absence of WeibelPalade bodies. Morphological and structural characteristics of transitional cells

Transitional cells morphologically resemble an intermediate composite of both epithelioid and mesenchyme-like cells (Fig. 1 C). Transitional MEC range in shape from elongated polygons to broad, bipolar cells. Intercellular contacts between transitional cells may range from focal associations to extensive, but discontinuous regions of plasmalemmal contact. Though transitional cells may migrate and exhibit lamellipodia at their anterior (leading) end (Fig. 4C), these cells do not form multilayers. Bipolar cells expressing transitional cell characteristics are normally present, encircling epithelial plaques in proliferating CAMP-supplemented cultures (Figs. 1A, 4C). The number of such cells is substantially reduced in confluent stationary-phase cultures. Transitional cells begin to reappear in confluent epithelial cultures within 5 h of exposure to CAMP-free media. Within 24 h of removing CAMP, transitional cells comprise the major cell population in the former epithelioid cultures. Transi-

Fig. 5 A-D. Fine-structural features of mesenchyme-like MEC. A In both transitional and mesenchymal MEC, regions of plasmalemma1 apposition do not exhibit the actin-associated dense bundles characteristic of epithelioid cells. B Transverse section through mesenchyme-like MEC. Dense assemblies of microfilaments (actin, see Fig. 4D) comprise apical-plasmalemma-associated stress fibers (arrowhea&). Bundles of intermediate filaments (vimentin, see Fig. C), running parallel to the stress fibers, are observed as stippled dots surrounding membranous organelles. C Fluorescence micrograph of FITC-conjugated vimentin filament matrix in mesenchyma1 MEC. Vimentin forms parallel linear arrays that run the length of the bipolar cells. D Tangential section through spindle-shaped MEC. Dense-staining actin stress fibers, subtending the apical plasmalemma, run parallel to the long axis of the cell. Narrow bundles of vimentin filaments, deeper in the cytoplasm, run parallel to the actin stress fibers

tional cells are derived from both the bipolar peripheral cells, as well as from former epithelioid cells that have contracted and assumed a more bipolar configuration. With continued growth in CAMP-free medium, the transitional cell population subsequently converts into mesenchyme-like spindle cells. Cell-cell contacts between transitional cells, though at times extensive, reveal no cytological specializations (e.g., the DPB). Weibel-Palade bodies are present but are generally fewer in number than in epithelioid MEC. Fluorescence assays of transitional cell cytoskeletons reveal that actin and vimentin matrices express qualities of both epithelioid and mesenchyme-like cells. The central reticular vimentin network characteristic of epithelioid cells undergoes dissolution and the remaining vimentin filament bundles generally run parallel to the long axis of the cell (Fig. 4B, C). Filamentous actin may be simultaneously present as both linear stress fibers and a dense encircling bundle (Fig. 4B). Transitional cells stained with FITC-conjugated antifactor VIII show the presence of fluorescent granular bodies within the cells. In addition, transitional cells deploy a factor-VIII-associated extracellular matrix (Fig. 4F). Effects of histamine on M E C cultivated in the presence and absence of C A M P

Histamine facilitates the conversion of epithelial MEC into the transitional phenotype. MEC presented with lo-’ M histamine after a 1 h incubation in CAMP-free medium rapidly transform into a transitional phenotype within 15 min. Transformation is associated with reorganization of the actin and vimentin cytoskeletons (Fig. 4B). CAMP inhibits the histarnine response, since no changes are observed in cultures fed histamine in medium containing CAMP. Endothelial cells continuously grown in histamine-supplemented CAMP-free medium exhibit a significant increase in proliferative activity as compared to that in CAMP-free control cultures. Hemocytometer counts of trypsinized cell suspensions reveal that after 7 days, lo-’ M histamine-supplemented medium induces a 200% increase in cell numbers over parallel cultures fed control (CAMP-deficient) medium. MEC maintained in histamine continuously express a transitional configuration and do not readily convert into the spindle-shaped phenotype. Histamine-treated transitional cells reexpress their epithelial phenotype upon refeeding with CAMP-supplemented growth medium. Consequently, transitional MEC do not represent an irreversible phenotypic modulation. EJfects of collagen on epithelioid andspindle-shaped M E C

When CAMP-associated epithelioid MEC are overlayed with collagen, most cells rapidly detach from the substrate and are lysed. The remaining epithelioid MEC aggregate into cell cords (Fig. 6). When the same experiment is carried out using spindle-shaped cells in medium

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Fig. 7A , B. Appearance of mesenchyme-like MEC in collagen gels. A Four days after MEC leave culture substrate and penetrate collagen gel, most cells acquire an extremely elongated spindle-shaped morphology characteristic of fibroblasts. B Mesenchyme-like MEC 6 days after application of collagen gel. When collagen gels are

layered over epithelioid MEC, former bipolar transitional cells frequently enter and migrate within the collagen matrix. In these cultures maintained in CAMP-containing medium, many of the migratory cells acquire a distinctive stellate appearance resembling that of perivascular dendritic cells in the dermis

without CAMP, the mesenchyme-like cells show no significant response to the collagen. Approximately 24 h after collagen deposition, mesenchyme-like cells begin to penetrate and migrate within the gel. Migrating cells assume either an elongated, spindle-shaped fibroblastlike morphology or a multibranched dendritic appearance (Fig. 7).

Transitional cells in histamine-treated cultures exhibit a mixed response when the cells are covered with collagen. Though some cells lyse and a few cell cords may form, the majority of cells show no response (Fig. 6). Within 24 h the remaining transitional cells begin to acquire a mesenchyme-like phenotype, and some of them begin to penetrate the overlying collagen matrix. Effects of y-irradiation on MEC transdifferentiation

Fig. 6A-F. Morphological appearance of epithelioid and mesenchymc-like MEC after layering collagen gels upon their apical surfaces. A, C , E Epithelioid MEC in CAMP medium. B, D, F Transitional cells induced with 2-h exposure to histamine (lo-’ M), interleukin-I (2 Ujml) and y-interferon (500 U/ml) in CAMP-free medium. A, B Five hours after layering collagen I gel over endothelial cells. Epithelioid cells (A) in CAMP-containing medium, initially resembling the culture illustrated in Fig. 1 A, show discrete morphological alterations. Cells lose junctional relationships and contract leading to dissolution of epithelial sheet. Transitional cells (B) already expressing junctional dissolution and some contraction (see Figs. 1 C and 4B) show minor morphological changes within 5 h of applying collagen gel. (Phase-contrast microscopy). C, D Within 12 h of layering collagen, former epithelioid cells (C) exhibit a dramatic morphological reorganization. Most cells have rounded up, with many organizing into elongated chains. Some cells have already bcen lysed, leaving behind granular debris (urrows). In induced transitional cell cultures (D), most of the MEC remain attached to the substrate as narrow, extended bipolar cells. Some of the cells still exhibiting epithelioid behavior have rounded up and been lysed ( u r r o ~ ~(Hoffman s). interference modulation rnicroscopy). E, F typical appearance of epithelioid MEC (E) 24 h after application of collagen gel. Viable MEC have aligned into cell cords. Most of the former epithelioid cells have lysed, leaving behind granular debris (arrows). Inflammatory mediator-induced transitional cells (F) remain essentially unchanged from their appearance 12 h earlier (see D). At this time transitional cells begin to detach from the substrate and penetrate the gel as mesenchymelike cells (see Fig. 7)

Within 8 h of y-irradiation and exposure to modified media the irradiated cells exhibit morphological changes identical to those occuring in nonirradiated control cultures. Irradiated cells grown in CAMP-free medium and medium supplemented with histamine (lo-’ M), interleukin-I (2 Ujml) or y-interferon (500 Ujml) acquired a bipolar transitional configuration within 12 h. Control and irradiated cultures were phenotypically indistiguishable (Fig. 8), although the latter eventually had significantly lower cell populations due to cell detachment. To identify the origin of modulated MEC in irradiated cultures, cell proliferation was assayed using a bromodeoxyuridine (BrdU)/anti BrdU peroxidase enzyme assay. In nonirradiated cultures, proliferating cells revealed nuclear uptake of BrdU. Irradiated cultures showed no evidence of BrdU incorporation. Consequently, the observed phenotypic modulation of irradiated cells in the absence of DNA synthesis reflects an example of direct transdifferentiation. Discussion Historically, histologists have defined endothelium as a specialized epithelial tissue [23]. Under homeostatic con-

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Fig. 8A-D. Response of control and inflammatory-mediator-treated cells to gamma-irradiation. A Epithelioid cells, similar to those illustratcd in Fig. 1 A, 24 h after irradiation. The endothclial cells remain essentially unchanged and maintain their epithelioid character. B, C, D Cultures fed either 2 h before or 2 h following irradiation with M histamine (B), 2 U/ml interleukin-I ( C ) or

500 U/ml y-interferon (D) in CAMP-free medium exhibit profound morphological alterations. Despite the irradiation treatment, which blocks cell division, modified MEC lose endothelial characteristics and acquire cytodifferentiative features of mesenchymal cells, such as Factor XIIla ( [ 5 9 ] , and Lipton, Bensch and Karasek in preparation)

ditions, the microvessel intima expresses the structural and functional characteristics of an epithelium. However inflammatory reactions compromise the epithelial integrity of the intima and may induce the expression of mesenchyme-related traits by MEC [ 31. Epithelial-mesenchymal differentiations reflect two dissimilar structural configurations that may be adopted by cells in multicellular animals [371. Epithelial tissues constitute coherent cellular sheets that line surfaces and cavities, while mesenchymal tissues are comprised of loosely woven networks of matrix-enmeshed cells [15, 16, 471. As coherent sheets of cells, epithelial tissues provide for the structural compartmentation characteristic of

multicellular organisms [47]. The primary function of an epithelium, such as the endothelium, is to serve as a selectively permeable barrier that both defines and regulates the composition of its contained compartment [15, 471. Epithelial barriers are fundamental in regulating the metabolism, respiration and homeostasis of an organism. Mesenchyme cells are derived from epithelia through epithelial-mesenchymal transformations [ 17, 18, 191. Differentiated mesenchymal derivatives provide structural and physiological support for their epithelium of origin. In this capacity, mesenchymal functions are concerned with the physical and nutritional support, shaping, motility and protection of the organism.

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Cultivated MEC us epithelium. This study, in conjunction with others [22, 26, 32, 551, reveals that microvessel endothelial cells in vitro, under normal growth conditions, express and maintain the characteristics of a true epithelium. Dermal microvessel endothelial cells form a cobblestone-patterned monolayer of squamous epithelioid cells when grown in media supplemented with cAMP [22]. Cultivated MEC synthesize type IV collagen, laminin and fibronectin and deploy them as a basal lamina [26, 27, 413. Organelles within the thickened central portion of the flattened MEC exhibit apical-basal polarized stratification, a defining characteristic of epithelia [47]. While membranous organelles generally exhibit apicalbasal polarization, the filamentous cytoskeletal organelles (i.e., actin and vimentin filaments) are deployed in a distinctive radially symmetric and concentric framework. In MEC, the actin and vimentin cytoskeleton, in association with the zonular junctional specializations resembles a complex terminal web. As in cuboidal and columnar epithelia, this web-like configuration may serve in supporting MEC epithelial functions [8]. The radial symmetry of the MEC vimentin matrix would presumably prevent cell disruption by facilitating a uniform distribution of lateral forces generated within cell sheets [8]. Contraction of the actin-rich dense peripheral bundle (DPB) would enable the endothelial cell to compensate for shape distortions generated by lateral tensions and surface-associated shear forces. In addition to providing a permeability barrier and supporting the cytoskeleton, epithelial zonular junctions are responsible for the maintenance of plasmalemmal domains and the regulation of intercellular diffusion properties [47]. Endothelial cells in vitro exhibit an apical-basal asymmetry in the distribution of plasmalemmal surface receptors and cell markers [7, 40, 561. In addition, microvessel endothelial cells in culture have been used as a model to assess the role of junctional specializations and intercellular cell adhesion molecules (ICAMs) in facilitating cohesive cell :cell binding phenomena [7, 361. Cultivated MEC further resemble epithelial cells with regard to their behavior in collagen gels. When embedded in type I collagen, the polygonal epithelioid cells detach from the substrate and are either lysed or reorganized into cell cords, which may cavitate, forming epithelial tubes and vesicles. This morphic response is a characteristic behavior pattern for ductile epithelial cells in vitro [14, 151. In these experiments, cells remaining adherent to the substrate initially express a transitional phenotype. Subsequently, these cells may acquire mesenchymal traits and penetrate the overlying collagen matrix. Cultivated MEC as mesenchyme. Collectively, the cytological features and functional behavioral patterns expressed by MEC grown in medium with cAMP reveal that dermal microvessel endothelial cells possess all the attributes of a true epithelium. When the same cells are grown in the absence of exogenous CAMP, each of the

epithelium-specific characteristics is supplanted by mesenchyme-specific attributes : (a) zonular junctions are disrupted; (b) apical-basal polarity is replaced by an anterior-posterior polarity; (c) endothelial cytodifferentiative expression is lost and reticuloendothelial traits are expressed; (d) synthesis of basal lamina components is terminated as cells begin to synthesize fibrous collagens ; and (e) the cells exhibit mesenchymal behavior in collagen gels. The first sign of impending MEC mesenchymalization following exposure to CAMP-deficient medium is the disruption of junctional contiguity. Subsequently MEC organellar distribution, formerly expressing an epithelial apical-basal polarity, undergoes reorganization to reflect an anterior-posterior polarity characteristic of migratory mesenchymal cells. Mesenchymal MEC exhibit an actin-vimentin cytoskeleton of parallel filaments expressing a bilateral symmetry around a cytological anterior-posterior polarity. This is a radical departure from the epithelioid cytoskeleton wherein filaments form a radially symmetric matrix around an apical-basal organellar polarity. Upon junctional dissolution, the apical plasmalemma loses its polarized epithelial character and expresses ICAM receptors that enable cell-cell binding on its former nonthrombogenic surface [33, 38, 441. These surface changes result in a loss of contact inhibition and lead to overlapping cell processes and formation of multilayered colonies [9, 571. The spindle-shaped MEC are irreversibly transformed in that they will no longer express epithelial traits if replated in CAMP-supplemented medium [57]. Expression of factor VIII antigen (von Willebrand factor), a specific cytodifferentiative feature of endothelial cell [45], is lost as MEC undergo mesenchymalization. Spindle-shaped MEC possess neither factor VlII antigen nor Weibel-Palade bodies [57]. The former intracellular factor VIII antigen, secreted by modified MEC, becomes incorporated as part of a fine extracellular network found enveloping the transitional and spindleshaped endothelial cells (Fig. 4 F). Recently, von Willebrand factor has been shown to possess endothelial integrin-binding receptor sites [ 1I]. Consequently the release of factor VIII antigen and its distribution as an extracellular network by modulated MEC may provide a scaffold for processes initiated by capillary injury, including hemostasis and the subsequent reactive role of these microvessel cells. The observed reticular organization of the factor-VIII-positive network closely resembles the glycosaminoglycan matrix secreted by cytokine-treated umbilical vein endothelial cells in culture [37, 381. Deposition of this matrix may be the consequence of an epithelial-mesenchymal transformation. While epithelial cells sit upon an extracellular matrix, mesenchyme cells are characteristically enmeshed within pericellular material. In addition to developing a factor VIII matrix, modified MEC also alter their extracellular milieu by profoundly changing their pattern of collagen synthesis. Spindle-shaped endothelial cells, true to their mesenchyme-like phenotype, synthesize collagen types I and

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I11 as compared to type IV collagen synthesis by epithelioid MEC [26, 27, 411. Spindle-shaped MEC behave as true mesenchymal cells in collagen gels. When collagen is layered upon mesenchyme-like MEC, the cells become slightly narrower but do not contract, detach or lyse. Beginning approximately 24 h after layering the collagen, mesenchymal MEC begin to penetrate and migrate within the gel as characteristic elongate fibroblast-like cells [ 191. Significantly, a subpopulation of these matrix-enmeshed cells acquire a stellate morphology and become structurally indistinguishable from perivascular dendritic cells present in inflamed dermis [54]. Transitional MEC. Five hours after exposing cultures to CAMP-deficient medium, MEC phenotypic modulation is first evident as intercellular gaps form in the squamous epithelial monolayer. However, mesenchymal spindle-shaped MEC only appear after a period of several days. In the interim, MEC express a transitional cell phenotype which exhibits epithelial as well as mesenchyma1 cytological features. Transitional cells are broad, flattened bipolar cells that make focal and discontiuous contacts with other endothelial cells. Transitional MEC exhibit a positive, yet reduced, staining for factor VIII antigen, an observation that correlates with their reduced number of Weibel-Palade bodies. Cells with transitional phenotypes are characteristically found encircling plaques of flattened MEC in actively proliferating cultures grown in CAMP-supplemented medium. Relatively few transitional cells are present in growth-inhibited confluent cultures. However, transitional cell types can be induced to reappear in such cultures following perturbations (e.g., wounding the monolayer) that produce a resurgence of cell division. The ratio of epithelial to transitional MEC is in part dependent upon the proliferative activity of the culture. Consequently, the cytodifferentiation of MEC in vitro normally includes a cell-cycle-related transitional phase. Epithelial MEC are dependent upon exogenous cAMP to maintain their phenotype. In the absence of CAMP, many epithelioid MEC contract and detach. Transitional cells are not appreciatively perturbed by exposure to CAMP-deficient medium. Therefore the observed reduction in cell numbers present 24 h after exposure to CAMP-deficient medium is directly related to the epithelial population at the start of the experiment. Interestingly, if histamine is added to CAMP-deficient medium the cultures exhibit a significantly greater number of attached cells after 24 h than do parallel cultures fed plain CAMP-free medium. Histamine, a potent mitogen [34], apparently induces epithelioid MEC to enter a stage of the cell cycle characterized by expression of a transitional cell phenotype able to maintain cell: substrate adhesion in CAMP-free medium. Transitional cells covered with collagen gels may express either epithelial or mesenchyme-like behavior. Most transitional cells remain as broad, bipolar cells adherent to the substratum when embedded in a type I collagen matrix. However, a substantial population of these cells may be lysed and some may even reorganize

into cellular cords. Within 24 h, some transitional cells may penetrate into the overlying gel and acquire a fibroblastic or dendritic mesenchymal phenotype. Contarninatirzg cell types and mesenchymal MEC origin. Cultures of microvessel endothelial cells derived from foreskin contain a small (< 5%) population of contaminant cells. The principal contaminants are readily distinguishable as melanocytes [22, 571. Since fibroblast growth is inhibited by CAMP,contaminating fibroblastic cells are reduced or eliminated with serial passages in CAMP-containing media [35]. MEC cultures used in this study, though passaged three to five times, may still contain a small contaminant population, Consequently, it may be questioned whether the transitional and mesenchyme cells are of endothelial or contaminant origin. Three observations confirm that the modified cell populations are primarily of endothelial origin, although contaminant cells may provide a minor contribution to the mesenchyme population. As reported earlier, transitional cells characteristically contain endothelial-specific Weibel-Palade bodies [57] and stain with antibodies against factor-VIII-related antigen. Secondly, large numbers of transitional cells or mesenchymal MEC appear within 24 h of refeeding with CAMP-free or cytokine-supplemented CAMP-deficient medium, respectively. Given that the mitotic cycle is greater than 12 h (Karasek, unpublished observations), the resulting large population of transitional and mesenchymal cells could not have been derived from a proliferating minor contaminant population. Lastly, y-irradiated confluent MECmonolayer cultures exposed to CAMP-free medium give rise to both transitional and mesenchymal cells in the absence of cell division. Second messengers and M E C phenotypic expression. The MEC epithelial phenotype can be induced directly with exogenous cAMP [2, 22, 551, by vasoactive agents like epinephrine or isoproterenol that employ cAMP as a second messenger [29] or by agents such as forskolin and cholera toxin that affect the synthesis of cAMP [2, 221. In contrast, preliminary studies reveal that histamine or other agents that utilize Ca2+ as their second messenger (e.g., norepinephrine, bradykinin, phorbol esters), induce dermal microvessel endothelial cells to alter their cytoskeleton and express an intermediate transitional cell configuration [29]. It is important to note that elevated levels of cAMP can block transitional cell formation by Ca2+-mediated primary signals. While sustained growth in CAMP-deficient medium induces MEC mesenchymalization over a period of several days, cytokines such as interleukin-1 or y-interferon bring about such changes within 24 h [37, 38, 44, 571. Preliminary studies reveal that cytokines not only induce cytological reorganization but also the expression of monocyte-related factor XIIIa [30].

MEC transifferentiation. Though the epithelial configuration of MEC might be considered to reflect a terminally differentiated state, the changeable nature of the MEC and their conversion into mesenchymal cells are not un-

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usual. Similar epithelial-mesenchymal transformations of differentiated tissues have been reported earlier [ 12, 17, 18, 611. Based on these studies, Hay [17, 18, 191 proposed the existence of master gene systems that control the expression of epithelial and mesenchymal phenotypes. Thus, dermal microvessel endothelial cells in vitro represent another epithelial-mesenchymal cell model. Unlike other models, MEC phenotypic modification is linked to CAMP expression, and during their modification, MEC can pass through a relatively stable intermediate transitional stage. As demonstrated by the y-irradiation study, the ability of MEC to alter their phenotype in the absence of a mitotic cycle convincingly confirms that transdifferentiation does not require cell replication. Our observations on MEC direct transdifferentiation are of profound theoretical significance for established concepts of gene expression in developmental biology [4]. Further, studies of environmentally mediated changes in gene expression that may take place in the absence of cell division in other organs offer alternatives to lineagerelated terminal differentiation [20]. MEC modification: Relevance to vessel iiitima in vivo. Homeostatic microvessel functions are mediated by epithelial MEC functioning as a selectively permeable membrane that regulates the exchange of molecular constituents between the blood and the surrounding tissue (Fig. 9A). Acute inflammatory responses or tissue injury lead to a local release of histamine by either mast cells or basophils, or even by endothelial cells themselves following induction of histidine decarboxylase activity [521. Histamine induces a transitional phenotype in affected MEC. The resulting morphological changes lead to intima1 disruption characterized by a loss of MEC junctional contiguity [6] and a cytological reorganization (Fig. 9 B). Endothelial cell retraction, intercellular gap formation and altered adhesivity of the MEC apical surface promote edema and leukocyte binding and emigration. In chronic inflammation, sustained elevated levels of histamine may induce a proliferation of transitional cells, similar to that occuring in culture. The increased population of transitional cells would account for the development of factor-VIII-positive spindle-shaped MEC observed near blood vessels in vivo [3]. Environmentally released cytokines may subsequently induce expression of mesenchymal cell properties (Fig. 9 B), including phagocytosis, fibrous collagen synthesis, and antigen presenting capabilities [33,44, 501. These mesenchymal properties, from an historic perspective, have been associated with inflammation and collectively considered as typical of cells of the reticuloendothelial system (RES, see [ 11). With the emphasis on studies of the role of circulating cells and the immune system in the inflammatory response, contributions of the endothelial cells to this process have essentially been disregarded in recent years. This view has become so narrow that the former RES is now referred to as the mononuclear-phagocytic system, a classification that ne-

Fig. 9. Schematic representation of proposed dermal microvessel modulation induced by inflammatory-related vasoactive amines and cytokines in vivo. E, epithelial cell; T, transitional cell; M , mesenchymal cell; L, leukocyte. A During growth, mitotic MEC transiently express transitional configuration. As cells reflatten they reacquire an epithelial phenotype. B In inflammatory reactions, MEC junctions are disrupted and cells assume a transitional configuration. This modulation provides for fluid leakage and leukocyte emigration. Following an acute inflammation, transitional cells may reexpress epithelial traits. However, under chronic inflammation, transitional cells may either directly transdifferentiate, or proliferate and subsequently express specific mesenchymal phenotypes as influenced by environmental factors (e.g., cytokines)

glects the potential contributions of MEC to the body’s defense mechanisms. Recent studies attributing phagocytic [48, 50, 571, immune-related [21, 30, 581, and myofibrogenic [13, 24,411 properties to modified microvessel endothelial cells provide a basis for reconsidering the MEC as a major participant in the inflammatory response. The MEC in vitro cell model lends itself to detailed analyses of various known and postulated factors that play a role in endothelial cell function and behavior in vivo. Using this MEC model, we have shown the wide range of cellular responses which endothelial cells are capable of displaying. The mesenchymal nature of some of these responses is conceptually challenging yet not surprising in retrospect. Endothelial cells appear to be broader in their function and in their ability to respond to perturbations than hitherto assumed. Vimentin us a mesenchyme-associated intermediate filament. The presence of vimentin intermediate filaments is considered diagnostic of mesenchyme cells, while certain cytokeratins are supposedly indicative of epithelial cells [ 14, 421. Since microvascular endothelial cells possess vimentin and not cytokeratins, it may be inferred that MEC are mesenchymal and not epithelial in nature. The concept of vimentin as a mesenchymal cell marker is troublesome in that there are a large number of notable exceptions. A variety of ectodermal epithelia ( e g , iris, lens, thymus, thyroid) and epithelial-like tissues derived from mesenchyme possess vimentin intermediate filaments [28]. In addition to the vascular endothelial cells, the mesothelium of the pleural, pericardial and

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peritoneal cavities, the germinal epithelia (Sertoli cells and follicular granulosa cells) and the perineuria all contain vimentin while simultaneously displaying epithelial phenotypes [42]. Historically, all of these tissues were formerly classified as endothelia, although current convention reserves this term exclusively for the vascular lining cells. Rather than classifying vimentin as a mesenchymerelated intermediate filament, it may be more appropriate to consider vimentin as an intermediate filament type present in any cell that is not in direct or indirect (i.e., via ducts or glands) contact with the external environment. Characteristically, vimentin-containing cells are completely enveloped within a matrix or ground substance (self, and have no exposure to a nonself environment. This may explain why cells that detach from kerdtin-containing epithelia may initiate synthesis of vimentin [14, 28, 421. Furthermore, any invasive tumor cell derived from a keratin-containing epithelium or any cell in tissue culture might also be expected to express a vimentin cytoskeleton. The restriction of vimentin-containing cells within environments recognized as “self” and the association of cytokeratin-containing cells with “non-self” interfaces may have significant implications in regard to the special immune characteristics of cells maintained within the lumens of virnentin-containing epithelia: blood cells, nerve cells and germ cells. Acknowledpents. The authors wish to thank Irma Daehne and Mary Jane Eaton for their excellent technical assistance. This work was supported by grants AR39470 and AR07422 from the National lnstitutes of Health and by grant N0014-89-J1098 from the Office of Naval Rcsearch.

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Microvessel endothelial cell transdifferentiation: phenotypic characterization.

Human dermal microvessel endothelial cells (MEC) have two basic functions: maintenance of tissue homeostasis and facilitation of inflammatory response...
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