extracellular matrix modulation of endothelial cell shape and motility ...

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WILLIAM C. YOUNG AND IRA M. HERMAN 1. Department of Anatomy ... Bovine aortic EC were seeded onto glass microscope coverslips that had been coated ...
jf. Cell Sci. 73, 19-32 (1985) Primed in Great Britain © The Company of Biologists Limited 1985

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EXTRACELLULAR MATRIX MODULATION OF ENDOTHELIAL CELL SHAPE AND MOTILITY FOLLOWING INJURY IN VITRO W I L L I A M C . Y O U N G A N D IRA M .

HERMAN1

Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, L'.SA.

SUMMARY

We utilized fluorescence microscopy and affinity-purified antibodies to probe the form and function of cytoplasmic actin in endothelial cells (EC) recovering from injury and grown on extracellular matrices in vitro. Bovine aortic EC were seeded onto glass microscope coverslips that had been coated with either BSA, fibronectin, type I and III (interstitial) collagens, type IV (basement membrane) collagen or gelatin. After EC that had been grown on glass, glass-BSA or extracellular matrix-coated coverslips reached confluence, a 300-400 fim zone of cells was mechanically removed to stimulate EC migration and proliferation. Post-injury EC movements were monitored with time-lapse, phase-contrast videomicrography before fixation for actin localization with fluorescence microscopy using affinity-purified antibodies. We found that the number of stress fibres within EC was inversely proportional to the rate of movement; and, the rates of movement for EC grown on glass or glass-BSA were approximately eight times faster than EC grown on gelatin or type IV collagen (X velocity = 0-5 fim/min versus 006/xm/min). EC movements on fibronectin and interstitial collagens were similar (X velocity = 0 2 /xm/min). These results suggest that extracellular matrix molecules modulate EC stress fibre expression, thereby producing alterations in the cytoskeleton and the resultant EC movements that follow injury in vitro. Moreover, the induction of stress fibres in the presence of basement membrane (type IV) collagen may explain the failure of aortic EC to migrate and repopulate wounded regions of intima during atherogenesis in vivo.

INTRODUCTION

In healthy blood vessels, endothelial cells (EC) function to provide a blood-compatible inner lining of the vessel wall (Zetter, 1981). Repeated injury to this squamous epithelial layer, resulting in a failure to maintain continuity between EC, may give rise to atherogenesis (Ross & Glomset, 1976). EC reside on and synthesize a basement membrane composed of numerous macromolecules, including collagens, proteoglycans and glycosaminoglycans (GAGs), as well as other glycoproteins (Hay, 1981). Because of this permeability barrrier, EC may depend upon diffusion of nutrients through the basement membrane for their normal metabolism. Thus, the contact and relationship between the EC abluminal surface and the basement membrane plays an important role in normal EC metabolism including the maintenance of intimal integrity. •To whom all correspondence should be addressed. Key words: anti-actin, endothelium, injury.

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In vivo, stability of the intima may be dependent upon the stress fibres of the EC cytoskeleton. Since stress fibres terminate in adhesion plaques that associate with the luminal and abluminal plasma membranes, these microfilament bundles may promote attachment of EC to the underlying basement membrane (Herman, Pollard & Wong, 1982; Wong, Pollard & Herman, 1983). For this reason the EC cytoskeleton has been proposed as a cellular mediator of the blood-shearing forces that occur at the luminal aspect of the intima in regions where blood velocity and turbulence are excessive (Dewey, Bussolari, Gimbrone & Davies, 1981; Herman e/ al. 1982). EC stress fibres in these areas are aligned parallel to blood flow and have recently been shown to modulate during states of hypertension (White, Gimbrone & Fujiwara, 1983) or following EC injury in vivo (Gabbiani, Gabbiani, Lombardi & Schwartz, 1983). Consequently, it seems likely that EC actomyosin serves to maintain intimal integrity by promoting EC-basement membrane adhesion via the plasma membrane, thereby anchoring the EC to the basal laminae of large vessels; but, modulation of the EC cytoskeleton occurs in response to vascular pathophysiology. A role has also been proposed for contractile proteins in the EC response to injury in vitro (Selden & Schwartz, 1979; Gottlieb, Heggeness, Ash & Singer, 1979). Removal of EC from confluent monolayers results in spreading, migration and proliferation (Selden & Schwartz, 1979; Scholey, Gimbrone & Cotran, 1977). The degree to which each of these types of behaviour is expressed is dependent upon the extent of injury inflicted in vitiv. The EC migratory response is dependent upon the form and arrangement of actin filaments, since cytochalasin B inhibits the post-injury migration of EC at the wound edge /';/ vitiv (Selden & Schwartz, 1979). Thus, an understanding of the EC contractile machinery present in migratory versus stationary cells may reveal how EC utilize contractile proteins to effect intimal stability and, or, post-injury motile force generation. Since EC stress fibres interact with the basement membrane of blood vessels via membrane attachment sites, molecules in the extracellular matrix may directly or indirectly effect the overall configuration of the cytoskeleton. We were interested to learn what influence extracellular matrix molecules might have on the overall arrangement of the EC cytoskeleton and whether this cell-matrix interaction could influence response to injury in vitiv. We used time-lapse, phase-contrast videomicrography to measure the rate and extent of living EC movements following injury as a function of extracellular matrix molecules, and characterized the form and distribution of cytoplasmic actin in the wounded monolayers using affinity-purified and labelled antibodies. Results of these experiments indicate that extracellular matrix molecules profoundly affect the rates of post-injury EC motility; and, coincident with these events there are significant alterations in the EC cytoskeleton.

Anti-actin localization in wounded endothelium

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MATERIALS AND METHODS

Antibody preparation Affinity-purified rabbit anti-actin immunoglobulin Gs (IgGs) were prepared against chicken gizzard (smooth muscle) actin as previously reported (Herman & Pollard, 1979). Non-adherent IgG fractions that did not bind to the affinity columns were taken as immune IgG not directed against actin and were used, as in the past, for control experiments (Herman & Pollard, 1979; Herman, Crisona & Pollard, 1981).

Endothelial cell isolation and preparation Bovine aortas were obtained from a local abbatoire on ice in Dulbecco's minimum essential medium (DMEM) cooled to 0 °C on ice. EC were isolated via collagenase irrigation as described by Jaffe, Hoyer & Nachman (1973) and transferred to 35 mm Petri dishes. EC colonies were isolated and cloned into 24-well plates. All EC clones were tested for factor VIII antigen by immunofluorescence using anti-factor VIII antibodies (Jaffeel til. 1973). A single clone of the cells between 5 and 10 passages was subsequently grown to confluence on either: (1) uncoated glass microscope coverslips; (2) glass coverslips coated with bovine serum albumin (BSA); (3) glass coverslips coated with fig quantities of bovine fibronectin (Biomedical Technologies); (4) glass coverslips coated with a dispersion of types I and III collagen (Ethicon Inc., Somerville, NJ); (5) glass coverslips coated with gelatin (Difco Lab., Detroit, MI); or (6) glass coverslips coated with type IV collagen obtained from bovine kidney cortex, a generous gift from Dr Sanyu Bixit (Nashville, T N ) . Concentrations of extracellular matrix molecules adsorbed to the glass coverslips were determined using the BioRAD protein assay and were found to be in the microgram range for all molecules tested (1-5 /xg/coverslip). Cells were grown attached to the coverslips submerged in DMEM supplemented with 5 % (v/v) calf serum buffered with 2-OmM-Hepes ( p H 7 3 ) .

Wounding of endothelial cell mono/avers EC monolayers were grown to confluence on glass, glass-BSA, fibronectin, collagen types I and III, gelatin and type IV collagen. Two days post-confluence, EC on various substrates were refed with DMEM plus 5% calf serum buffered with 2-0mM-Hepes (pH7-3). A 300-400 fim wound was mechanically inflicted in each monolayer using a sterile scalpel blade. The sterile procedure was performed under observation using an inverted Olympus microscope equipped with phase-contrast optics.

Quantitative analysis of endothelial cell movements following injury in vitro The wounded cell monolayers attached to the coverslips were placed in a 35 mm Petri dish, submerged in growth media, sealed with parafilm to maintain an atmosphere containing 5 % COj, and placed onto the temperature-controlled (37 °C) stage of an Olympus light microscope equipped with interference and phase-contrast optics. A microscope-interfaced MTI-Dage 65 TV camera, coupled to an NEC video tape recorder with built-in time/date generator was used to record living EC movements at the wound site. EC movements were recorded for 3 h following injury, at which time the cells were removed from the microscope stage and prepared for fluorescent antibody staining. For analysis of EC post-injury movements a scaled grid from the camera image of a stage micrometer was used to make a vinyl overlay for the monitor. Thirty to fifty cells from each run of every experimental group were tracked by single frame analysis. The positions of nuclei and leading edges were marked and instantaneous velocities and net movements plotted. We did this for cells as deep as eight rows away from the wound edge where directional movement was nil.

Preparation of endothelial cells for fluorescent antibody staining Each wounded monolayer was prepared for fluorescent antibody staining using formaldehyde fixation followed by acetone extraction as described previously (Herman el til. 1981).

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Staining ofwounded endothelial cell monolayers with affinity-purified anti-actin The fixed monolayers of aortic endothelium were incubated with a 12/u.g/ml solution of purified anti-actin for 60min at room temperature. Each monolayer was then washed in 100 ml of phosphate-buffered saline (PBS = 0015 M-sodium phosphate, pH7-5, 0-15 \i-NaCl) three times for S min. After buffer equilibration, fixed and permeabilized cells were incubated in 50/xg/ml of rhodamine-labelled goat anti-rabbit IgGs (Cappel Labs), for 60min at room temperature. Each monolayer was again rinsed three times for 5 min in PBS. Each coverslip was then mounted onto a glass microscope slide using a 9:1 (v/v) dilution of glycerol: PBS and scaled with nail polish.

Photomicrography Fixed and stained EC monolayers were observed in phase-contrast and fluorescence microscopy using a Zeiss Photomicroscope III equipped with the appropriate narrow band-pass barrier and excitation filters needed for rhodamine fluorescence. The microscope is equipped with a 40X planeofluor (NA 1-0) and a 63x planapochromat objective lens (NA 1-4). For photomicrography, Tri-X negative film was rated as ASA = 1000 and developed in Acufinc Developer for 4-75 min at 20 °C.

Quantification of endothelial cell motility and stress fibre expression Living cells. The time-lapse video tape recording of EC motility following injury was used to measure the rate and extent of EC motility following wound infliction. An image of a stage micrometer was projected onto the TV screen in order to scale post-injury migration. Fixed cells. After fluorescence micrography of fixed and stained cell cultures was complete, the negatives were projected onto a screen at XC80. Several parameters of EC stress fibres were then measured. Fibre width was measured at the mid-point of each stress fibre with calipers. Fibre length was also measured, in the same clearly disccrnable stress fibres. A scaled grid made up of 100(im2 units was used to measure stress fibre density. The number of fibres appearing within a 100fim2 area was digitized for cells grown on each substrate. A count of total fibres per cell (;; > 420) was also performed. Cell size was determined, as a function of surface area, by computing mean cell width and length for cells in which fibres were counted. A statistically significant number of EC cultures grown on different substrates were analysed (/; = 24).

RESULTS

We examined the influence of various extracellular matrix molecules on the motility of EC following injury. Using time-lapse video micrography to monitor the rate and extent of living EC movements and fluorescent anti-actin staining of these same migrating cells after fixation, we were able to correlate rates of motility with specific actin arrangements. Our results indicate that: (1) post-injury EC movements are modulated by extracellular matrix molecules; (2) the distribution of actin within the cytoplasmic matrix of wounded EC monolayers is profoundly affected by varying the composition of the extracellular matrix; and (3) the density of stress fibres in EC cytoplasm varied inversely with the velocity of the post-injury migration. Effect of extracellular matrix molecules on endothelial cell motility Time-lapse video micrography proved to be an excellent method to monitor the movements of EC remaining in wounded monolayers. Cell movements were recorded for 3h following injury, before fixation and antibody staining.

Anti-actin localization in wounded endothelium

Fig. 1. EC migratory response to injury in vilm. Phase-contrast micrographs (A) and fluorescent micrographs ( B - D ) of bovine aortic EC cultured on glass. After mechanical wounding, cells migrate into the denuded region (A). Motile cells denoted in \ as • and * are shown in B and C after staining with fluorescent anti-actin. Note the diffuse fluorescence and relative lack of stress fibres in these migrating cells. Arrows in c indicate prominent advancing pseudopodia. Cells that have repopulated the wound (within 150min after injury) are shown in o. A, X180; H-D, X530.

When bovine aortic EC were grown on glass or glass-BSA substrates (Fig. 1), mechanically induced wounds in the monolayer precipitated characteristic cell movements. EC at the immediate wound edge extended a variety of lamellar and pseudopodial cytoplasmic processes into the wounded region; and, with time, these cells became polarized, often with their long axes oriented normal to the wound edge. These cells exhibit directed migration. Once cell-cell contact was disrupted due to the probing movements of an EC moving into the wound, the adjacent cells moved forward and, or, laterally into the space vacated by its neighbour. 'Phis

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Fig. 2

Anti-actin localization in wounded endothelium

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movement slowed down considerably and became increasingly less directional in EC further away from the wound edge. Cellular movements, which appeared as random, rapid surface blebs in time-lapse, were witnessed up to eight cell rows back from the wound. EC along the wound edge, which were cultured on glass, exhibited an average post-injury migration rate of 05/i.m/min. EC move most quickly directly after removal of neighbouring cells (2-0/u.m/min) with little directional movement once cell-cell contact is established. EC two and three cells deep (away from the wound edge) exhibited rates of 0-25 and 0-12/u.m/min, respectively. These monolayers (grown on glass or glass-BSA) were the only EC in our study capable of re-covering the denuded region during the 3 h between injury and fixation. The aortic EC cultures grown on various extracellular substrates expressed varying rates of post-injury migration. Cell morphology also varied according to the rate of cell movement. EC cultured on fibronectin and collagens I and III (Fig. 2) migrated into wound sites at approximately equivalent rates (02/Lim/min). Cells grown on glass moved roughly 2-5 times faster than the cells that were cultured on fibronectin or interstitial collagens. EC grown on these substrates rarely extended spindle-shaped processes into the wound and the membranes exhibited less ruffling, seen by observing the injury site in time-lapse. EC cultured with fibronectin, and collagens I and III, which were two and three cells deep from the wound edge, moved at rates of 0-06 and 0-04/u.m/min, respectively. When EC monolayers were grown on gelatin or type IV collagen (Fig. 3) and then injured, the cells at the wound edge moved at a rate of 0-07 and 0-06/xm/min, respectively; a net movement that was eight times slower than EC cultured on glass (see Table 1). The EC grown on basement membrane collagen and gelatin also had the most regular, uninterrupted borders. These cells were flattened, spread out and very rarely exhibited long, cellular extensions perpendicular to the wound edge. Motility of these EC populations was restricted to membrane ruffling at the free edge along the wound site. Effect of extracellular matrix molecules on cytoplasmic act in arrangement After the bovine aortic EC had been allowed to recover for 3 h following injury, the monolayers were fixed and stained with anti-actin. Measurements of cell size (surface area), stress fibre length, stress fibre width, and stress fibre density (number) were made from anti-actin-stained EC grown on different substrates. When EC were grown to confluence on glass, injured, and then stained with fluorescent anti-actin 3h later, two distinct fluorescent patterns were observable: one component was fibrous, and the other was diffuse. Bright, marginal diffuse Fig. 2. Post-injury analysis of bovine aortic EC cultured on fibronectin and interstitial collagens. Areas at the wound edge ( * and *) seen with phase-contrast microscopy (A,IJ) are seen in B with prominent fluorescence indicating membrane ruffling (arrows), c demonstrates the typical fluorescent actin localization seen in EC at the wound edge when grown on fibronectin. Similar results are obtained when aortic EC are cultured on interstitial collagens ( D - F ) . Note the numerous stress fibres seen in H,C,I:,F. \, X400; B-C, X695; D, X135; E - F , X625.

W. C. Young and I. M. Herman

Fig. 3. Influence of collagenous components on EC migratory response to injury. Phase-contrast (A) and fluorescence ( B - C ) micrographs of EC cultured on type IV collagen. Cells shown at the wound edge in A as * are seen in 11 exhibiting numerous fluorescent stress fibres. Similar fluorescent patterns are seen in EC at the edge of a denuded region in D when gelatin is used as the extracellular matrix. \, x 170; 11, X530; C - D , X750.

Anti-actin localization in wounded endothelium

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Table 1. Effects of extracellular matrix on post-injury motility and stress fibre expression in cultured aortic endothelial cells

Glass Fibronectin Collagens I, III Gelatin Collagen IV

Stress fibre density* (no. fibres/100 /xm2 field)

Endothelial cell motility (fim/min)

1-08 1-74 1-90 2-41 2-71

5-1 x 10"' 2-2 X 10"' 1-7x10"' 0-7 X 10"' 0-6 X 10~'

• For each group: no. cells at wound edge used in analysis = 56< x < 105; no. 100/xm2 fields used in analysis = 390 < X < 725: total no. fibres counted within individual 100 fim2 fields = 422