Chlorpromazine modulates the morphological macro- and microstructure of endothelial cells

I. S. Hueck1, H. G. Hollweg2, G. W. Schmid-Schönbein3, and G. M. Artmann1

1 Cell Biophysics, University of Applied Sciences Aachen/Juelich; 2 Institute of Pathology, Rheinisch Westfälische Technische Hochschule Aachen, Neues Klinikum, Germany; and 3 Department of Bioengineering, University of California San Diego, La Jolla, California


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chlorpromazine (CP), an amphipathic, antipsychotic agent, causes concave membrane bending in red blood cells with formation of stomatocytic shapes by modulation of the phospholipid bilayer. This study was designed to investigate the effects of CP on the shape of bovine aortic endothelial cells (BAEC) and their membranes in confluent monolayers with phase-contrast and transmission electron microscopy. Exposure of BAECs to nanomolar levels of CP leads to membrane curvature changes. With increasing CP concentrations, the membrane assumed a shape with enhanced numbers of intracellular caveolae and projection of pseudopodia at all junctions. At higher CP concentrations (up to 150 µM), the endothelial cells assumed almost spherical shapes. The evidence suggests that CP may affect lipid bilayer bending of BAECs in analogy with previous observations on erythrocytes, supporting the formation of caveolae and pseudopodia in BAECs due to the induction of concave membrane bending, as well as an effect on endothelial cell membrane adhesion at higher CP concentrations with loss of cellular attachment at junctions.

membrane vesiculation; pseudopodia formation; phospholipid bilayer bending; endothelial cell culture; caveolae


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RECENT EVIDENCE INDICATES that endothelial cell plasma membrane vesiculation in the form of caveolae serves as a membrane reservoir during stretch of the endothelial cells and possibly for transendothelial transport in certain domains of the cytoplasm (16, 24, 26). Previous studies have shown that the amphipathic, cationic, and antipsychotic drug chlorpromazine (CP) modulates the phospholipid bilayer balance by intercalation into the inner leaflet of the red blood cell plasma membrane (4). The drug may reach both sides of the membrane because it can cross the membrane, but, thereafter, CP selectively inserts into the inner half of the membrane bilayer by virtue of its cationic properties (1, 4). According to the bilayer couple hypothesis (28), one-sided intercalation leads to expansion of the inner membrane layer relative to the outer layer causing the red blood cells to adopt a cupped shape. Because CP accumulates at the inner leaflet of the membrane bilayer, it may also modulate the activity of membrane-associated enzymes, located in the inner half of the bilayer, that are involved in phosphoinositide turnover (4, 27). By changing the membrane environment and the surface charge density (1, 14), the inward shift of phospholipids occurs faster than the diffusion outwards (27). Thereby, CP induces a new bilayer asymmetry and an inner-layer expansion that causes a bilayer buckling without changing the membrane protein composition or affecting ATP levels (4). Thus CP causes a time- and concentration-dependent concave plasma membrane bending that leads to macroscopic morphological changes in red blood cells toward stomatocytic cell shapes (2, 12, 18, 21). These shape changes are associated with relatively large radii of curvatures in micrometers. Smaller radii of curvatures induced by CP, however, were also observed in red blood cell membranes by Haegerstrand and Isomaa (13). At concentrations above ~25 µM, CP induces membrane lipid scrambling and formation of endovesicles with a diameter of ~150 nm (13).

Although there is evidence that drugs that affect cholesterol distribution serve to influence the formation of caveolae (26), the effects of CP on the endothelial cell morphology is unexplored. In this study, we investigate the possible effects of CP on plasma membrane curvature in endothelial cells. First, we investigated from a wide range of CP concentrations the macroscopic morphology of endothelial cells using phase-contrast images. In the second part, membrane curvatures at nanomolar CP concentrations were studied using transmission electron microscopy (TEM). In addition to membrane curvatures and caveolae, the formation of pseudopodia was examined.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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EC Cultures

Bovine aortic endothelial cells (BAEC) were isolated from fresh calf aortas according to the method of Booyse et al. (3) and cultured according to Zink et al. (31). Subcultures were obtained by removing the cells with trypsin solution (0.5 mg/ml) from the bottom of the well, centrifugation at 100 g for 10 min, and replating (dilution 1:7). Cells of the same culture at passage 9 were used in all experiments. The cells were plated on glass slides pretreated with 1% gelatin solution for 30 min at room temperature. Subcultures had been grown for 8 days in DMEM (Sigma) containing FCS at a final concentration of 10%, 0.2 mg/ml penicillin, 0.2 mg/ml streptomycin, and 0.48 mg/ml L-glutamine in an incubator with 5% CO2-95% air atmosphere at 37°C. Growth of the cultures was examined daily until confluent monolayers (100,000 cells/cm2) were obtained.

Drugs and Materials

Immediately before the start of the investigations, the cells were washed carefully in Dulbecco's PBS (9.6 g/l, Sigma) containing 0.1% glucose adjusted to pH 7.4 and 310 mosmol/kgH2O. Afterward, cells were treated with CP solutions in PBS for 15 min.

CP hydrochloride (C17H20Cl2N2S, molar weight 355.3 g, water soluble; Sigma) is used in clinical applications for psychotherapeutic depressant actions on the central nervous system (22). At concentrations below 200 µM, it does not significantly affect the osmolarity or the pH of a PBS (22). The CP concentrations used on the cell cultures were between 5 and 150 µM for phase-contrast investigations and between 1 nM and 10 µM for the TEM studies.

Image Analysis

The overall shapes of living BAECs were analyzed by phase contrast at ×200 magnification (Zeiss Axioplan). The focal plane of the microscopic image was adjusted using an automated autofocus routine (2, 25).

During treatment with CP, the cells were kept at 37°C in a controlled water bath and their image was recorded by a video camera (type NC-67M, Dage-MTI) and stored on VHS cassettes. Images were digitized for a period of 15 min of CP treatment because there occurred no further significant shape changes after this period of time (data not shown).

Ultrastructural cell shapes were examined by analysis of TEM images at magnifications between ×3,000 and ×50,000 (Zeiss EM 902). Immediately after the 15 min CP treatment, cells were fixed with 1.5% glutaraldehyde (Boehringer Ingelheim) in PBS (310 mosmol/kgH2O, pH 7.4) for 1 h. The use of isotonic fixation served to minimize tissue shrinkage during fixation. After fixation, the cells were washed in PBS three times for 15 min and postfixed in 1% osmium tetroxide (Boehringer Ingelheim; 310 mosmol/kgH2O, pH 7.4) for 1 h. Specimens were washed in distilled water, dehydrated in multiple steps of increasing concentrations of ethanol, and embedded in Reale's Epon embedding media (Boehringer Ingelheim). The Epon blocks were placed on the glass slides with the cells and polymerized for 8 h at 37°C followed by 56 h at 60°C. After polymerization, the cell monolayers on the bottom of the Epon blocks were carefully detached from the glass slides. To completely surround the cells with Epon on both sides, the blocks were trimmed into small rectangles and embedded for a second time using the same Epon embedding procedure as described above.

The final Epon blocks were cut into 1-µm-thick sections for light microscopy and silver color sections (~700 Å) for electron microscopy using a microtome (Reichert Ultracut S, Leica, Germany) with diamond knives. Thick sections for light microscopy were stained with Methylene Blue to control cell layers. Thin sections for electron microscopy were stained with uranyl acetate (2 g/100 g) and lead citrate (1.3 g/100 g) in a grid-staining instrument (LKB Bromma, Leica).

Cell Shape Indices

Two indices were used to analyze the topical phase-contrast view of the cell morphology: 1) the relative cell area and 2) the area circularity index. The relative cell area is defined as cell surface area after CP treatment divided by control area of the same cell before CP treatment. This index serves to detect changes in cell spreading and area of attachment. The cell surface area circularity index (ACI) is defined as a dimensionless ratio of the square of the cell area perimeter length (P) divided by the cell surface area (A) (Fig. 1)
ACI = <FR><NU><IT>P</IT><SUP>2</SUP></NU><DE><IT>A</IT> × 4&pgr;</DE></FR> (1)
The ACI is 1 for a circular cell and it is >1 in noncircular cells. The ACI serves to detect changes in cell stretching and cell sphericity and was used in previous studies to define cell shapes (6). The ratio of perimeter squared to area was normalized by 4pi so that the ratio has a value of unity for a circle.


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Fig. 1.   Illustration of area circularity index (ACI) for phase-contrast images of bovine aortic endothelial cells (BAEC) at ×250 magnification. Square of perimeter line length (P) divided by cell surface area (A) defines dimensionless ACI as an index for sphericity of cells.

To analyze electron microscopic images, which provide single-section views of the cells, the ACI and the perimeter regularity index (PRI) were used. First, the ACI was used to detect a change in the cell sphericity in a side view and, therefore, a possible change in the cell's detachment from the extracellular matrix. Second, to quantify caveolae and pseudopodia within the plasma membrane, the membrane PRI was used. The PRI is defined as a dimensionless ratio between the difference of true membrane length (including caveolae) and a straight-line approximation of the same cell and the straight-line length (Fig. 2)
PRI = <FR><NU>true membrane length − straight line length</NU><DE>straight line length</DE></FR> (2)
The PRI is 0 in cells without caveolae. All cell morphological measurements were obtained with an image analysis software (modified Optimas 4.1, Bio Scan).


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Fig. 2.   Illustration of perimeter regularity index (PRI) for transmission electron microscopy (TEM) images of BAEC at ×25,000 magnification. PRI is dimensionless ratio representing relative change between true membrane length (including caveolae) and straight-line approximation. It is an index of cell surface irregularity.

Statistics

The results are presented as the mean (±1 SD) of (n = 5) different cells at each CP concentration. The phase-contrast measurements were repeated three times with different cell subcultures of the same passage.


    RESULTS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Phase-Contrast Images

Projected cell surface area. With increasing CP concentrations, a progressive decrease of the projected cell surface area was encountered (Fig. 3) down to 25% of control values at the highest CP concentration of 150 µM (Fig. 4). The reduced cell spreading was accompanied by opening of gaps along the junctions of the endothelial cells. Eventually, the endothelial cells completely detached from one another (Fig. 3).


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Fig. 3.   Phase-contrast images (×200 magnification) of cultured BAEC exposed for 15 min to stepwise increasing chlorpromazine (CP) concentrations. Compared with control (a), cells opened some intercellular gaps at 10 µM CP (b) but retained their stretched shapes. With rising CP concentrations (c-e), cell sphericity increased and intercellular gaps widened, finally abolishing intercellular attachment completely at 150 µM CP (f).



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Fig. 4.   CP induced decrease in projected cell area observed in phase-contrast images. A steady decrease >10 µM CP was encountered down to a baseline of 25% of control values at 83 µM CP and above.

Area circularity index. At CP concentrations below 10 µM, the ACI increased with rising concentrations (Fig. 5). The maximum ACI was observed at ~10 µM at a level of ~130% of control values, marking a peak in the degree of cell stretching. At concentrations between 18 and 83 µM CP, the ACI linearly decreased, and at concentrations >83 µM CP, almost spherical cell shape was observed. These data indicate that concentrations of CP significantly above therapeutic levels (~0.1 µM blood plasma levels) affect the intercellular junctions as well as the attachment to the extracellular matrix. Nanomolar concentrations of CP up to 1 µM did not reveal effects detectable with phase-contrast imaging (data not shown).


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Fig. 5.   CP induced effects on ACI observed in phase-contrast images. ACI increased <10 µM CP with rising concentrations to maximum of 130% at 10 µM CP. It linearly decreased between 18 and 83 µM CP, leading to an almost spherical cell shape at concentrations >83 µM CP.

Transmission Electron Microscopy

Area circularity index. Cells treated with <5 µM CP did not show a change of the ACI values measured on TEM images (Fig. 6). At 10 µM CP, the ACI was significantly decreased, indicating the onset of a change in cell shape toward a more spherical form. These data suggest that CP significantly affects cellular junctions above concentrations of 5 µM.


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Fig. 6.   Effects of CP on ACI observed in TEM images. Below 5 µM CP, ACI remained unchanged at control levels. At 10 µM CP, ACI significantly decreased, indicating beginning of a change in cell shape toward a more circular form.

Perimeter regularity index. Although a change of the ACI at nanomolar CP concentrations was not observed, ultrastructural curvature changes of the cell membrane occurred, as PRI data showed. As the PRI values indicated, ~10% of the total membrane line length in control cells was involved in caveolae formation. At rising CP concentrations, the PRI increased as high as eight times that of control (Fig. 7, measurements of the total cell membrane length). The membrane developed more intracellular caveolae accompanied by an increased tendency to form vesicle chains and an increasing number of large-sized vesicles most prominent at 5 and 10 µM CP (Fig. 8, membrane sections as example). The drastic increase in PRI between CP concentrations of 0 and 5 µM (Fig. 7) was accompanied by no significant change in ACI (Fig. 6). Thus the PRI represents more sensitively tiny changes in the caveolae formation than the ACI. The ACI is a two-dimensional measure from a projected image of the three-dimensional cell. There exists no simple mathematical relation between the projected cell area and the actual three-dimensional geometry of the cell, except when the ACI has a value of unity for a circle. Only in this exceptional case it may be assumed that the simple relation between the projected circular area and a spherical cell shape due to cortical stress acting on the cell's surface (24) applies. However, a spherical cell shape cannot be completed when the cell is still attached to the surface, which was seen up to 150 µM CP (Fig. 3f). Enhanced formation of pseudopodia was observed along the cell periphery, predominantly at intercellular junctions between endothelial cells (Fig. 9).


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Fig. 7.   CP induced effects on PRI observed in TEM images. PRI control values showed that ~10% of total membrane length was involved in caveolae formation. At rising CP concentrations, PRI increased as high as 8 times that of control, indicating ultrastructural shape changes of cell membrane.



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Fig. 8.   Examples of caveolae formation in membrane of cultured BAEC when exposed for 15 min to rising CP concentrations (TEM images at ×25,000 magnification). Membrane developed more intracellular caveolae accompanied by an increased tendency to form vesicle chains most prominent at 5 and 10 µM CP.



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Fig. 9.   Pseudopodia formation in cultured BAEC after exposure to 5 µM CP for 15 min (TEM images at ×13,500 magnification). Enhanced formation of pseudopodia was observed along cell periphery. In perinuclear cell area, smaller pseudopodia were formed, but most prominent was formation at intercellular junctions in distal cell area.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Erythrocytes undergo shape transformation to spiculated cells (echinocytes) or cupped cells (stomatocytes) in response to various treatments (2, 21), including exposure to amphiphilic compounds (4, 18). The action on cell membranes of a cationic amphipath, like CP, is predictable according to the bilayer couple hypothesis (28) and has been repeatedly observed in red blood cell membranes (2, 9, 12). High-lipid bilayer bending in the form of endovesiculation induced by cationic amphiphiles has been described by Haegerstrand and Isomaa (13). The fact that a wide variety of amphipaths causes vesiculation in erythrocyte membranes indicates that vesiculation is a generalized characteristic of a membrane state (4). The evidence provided in this study demonstrates that CP does affect the endothelial cell membrane by generating membrane vesiculation, caveolae, and pseudopodia.

At elevated CP concentrations, cell detachment and retraction into spherical shapes occurred. Cell and membrane relaxation, due to a loss of cellular attachment at junctions, must be involved. This may indicate a CP-induced initiation of cell death as an apoptotic process (5). Phospholipid bilayer modulation, as observed in red blood cells (2, 27), may contribute to the loss of attachment via bilayer bending and lipid scrambling affecting the adhesion at junctional complexes (30).

At nanomolar concentrations of CP, the results suggest that the asymmetric CP interaction with the bilayer leaflets may influence the lipid bilayer curvature (11) and induce endovesiculation. This hypothesis is supported by the fact that at nanomolar concentrations, the cells remained attached at junctions, and no overall cell shape changes were visible in the phase-contrast images. In analogy to Haegerstrand and Isomaa's (13) observations in red blood cells, CP induced formation of caveolae at the endothelial cell membrane with almost no variation in size of the vesicles below 10 µM CP concentrations. With increasing CP modulation of the membrane bilayer, a rising number of caveolae of the same size between 700 and 800 Å appeared. According to the Laplace equation for membranes, a congregation of larger vesicles is to be expected, but almost no larger caveolae formations were observed. It is possible that the cytoskeleton is preventing the formation of larger caveolae. The cytoskeleton may be important in stabilizing the membrane shape, but the influence of CP on the remodeling of the cytoskeleton remains to be explored.

CP interacts with phospholipid membranes, produces changes in membrane fluidity, and modifies membrane permeability (1, 14). It is possible that an area imbalance between the two monolayers in the lipid bilayer of the membrane, as discussed in red blood cell shape changes (2, 9, 10, 11), may promote vesiculation in local domains of endothelial cells as well. Local regions of the membrane in the resting state may have high-membrane curvature of the order of plasmalemmal vesicles (24). By loss of tension through the cytoskeleton, the membrane may relax to its resting state with high-membrane curvatures. The resting membrane state may form similar-sized vesicle shapes with an increase in bending moment in the membrane. Therefore, remodeling of the lipid bilayer molecules through agents like CP can lead to shape changes even when the cells are still attached at cellular junctions (7, 8, 17, 23). Furthermore, no change in the membrane protein composition had been found in comparison of membrane domains with and without vesicles (26).

The shift in endothelial cell membrane vesiculation in the presence of CP may influence transendothelial transport in vivo (24, 29). Further investigations have to be carried out to elucidate the mechanism for caveolae formation as a result of phospholipid bilayer modulation by experimentally changing the endothelial cell membrane composition using other substances, like cholesterol (19). Furthermore, different methods for three-dimensional visualization of vesicle shapes (15, 20, 23) may provide further leads to these questions.

The generation of pseudopodia in endothelial cells induced by CP may also influence the hemodynamic resistance in vivo, especially in small blood vessels (16). Further in vivo studies with CP are required to examine these questions in more detail.

There exist a variety of membrane-active compounds that affect the lipid bilayer bending (11) by inducing either concave or convex membrane curvature (9). Some of them are used for microcirculatory therapy (10). We expect that these substances modify the microstructure of endothelial cells and may influence the transcellular transport as well as blood cell-endothelial cell adhesion. The current findings, thus, represent a first approach to shed light on the relationship between membrane curvature of endothelial cells and lipid bilayer modulation induced by amphipathic compounds.


    ACKNOWLEDGEMENTS

We thank C. Sturm and Prof. C. Mittermayer, Institute of Pathology, University Klinikum, Rheinisch Westfälische Technische Hochschule Aachen, Germany, for assistance in the preparation of the TEM specimen and G. Zischke, Aachen University of Applied Sciences, Germany, for instructions for the use of the electron microscope. Many thanks for useful recommendations to Dr. Ursula Sahm, Aachen University of Applied Sciences, Germany, to Frank Delano at the University of California San Diego, as well as to our colleagues in the Dept. of Cell Biophysics, Aachen University of Applied Sciences, Juelich, Germany, for technical support and consultation.


    FOOTNOTES

This work was supported by a grant from the Ministry of Science and Education North Rhine Westfalia (to G. M. Artmann) and a Deutscher Akademischer Austauschdienst stipend (to I. S. Hueck).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: I. S. Hueck c/o G. M. Artmann, Dept. of Cell Biophysics, Aachen University of Applied Sciences, Ginsterweg 1, 52428 Juelich, Germany (E-mail: ARTMANN{at}FH-Aachen.de).

Received 28 December 1998; accepted in final form 10 December 1999.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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