Cocaine-Induced Increase in the Permeability Function of Human Vascular Endothelial Cell Monolayers

Experimental and Molecular Pathology 66, 109–122 (1999) Article ID exmp.1999.2253, available online at http://www.idealibrary.com on

Cocaine-Induced Increase in the Permeability Function of Human Vascular Endothelial Cell Monolayers1

Frank D. Kolodgie, Patricia S. Wilson, Wolfgang J. Mergner,* and Renu Virmani2 Department of Cardiovascular Pathology, Armed Forces Institute of Pathology, Washington, DC 20306-6000; and *Department of Pathology, The University of Maryland, Baltimore, Maryland

Received December 14, 1998; accepted February 10, 1999

The effects of cocaine on endothelial cell macromolecular transport, electrical resistance, and morphology were assessed. In confluent endothelial monolayers grown on microporus filters, cocaine (0.01 to 1 mmol/L) induced a rapid concentration-dependent increase in permeability to peroxidase and low density lipoprotein. Along with increased transport, the cocaine effect was paralleled by a decrease in transendothelial electrical resistance. Alterations in membrane resistance were fully reversible following washout of the drug, providing evidence that cocaine does not cause permanent injury to the integrity of the monolayer. Cocaines major metabolites, benzoylecgonine and ecgonine methyl ester, had minimal effect on electrical resistance properties, whereas monolayer impedance was markedly depressed by the novel cocaine/alcohol metabolite, cocaine ethyl ester (cocaethylene). Morphologic studies of cocaine-treated endothelial cells revealed a marked disruption of F-actin and the formation of intercellular gaps; no evidence of cell lysis and/or detachment was noted. Forskolin, a potent activator of adenylate cyclase known to promote the endothelial cell barrier function, impaired cocaine-induced changes in electrical resistance and morphology. Cocaine, however, had no effect on resting levels of intracellular adenosine 38,58-cyclic monophosphate (cAMP) in confluent endothelial monolayers. In summary, the results indicate that cocaine directly induces structural defects in the endothelial cell barrier which enhance the transport of macromolecular tracers, the mechanism does not appear to involve intracellular cAMP. q 1999

Key Words: cocaine; endothelium; permeability; forskolin; low-density lipoprotein; peroxidase.

INTRODUCTION

The acute coronary syndromes associated with chronic cocaine abuse in young, otherwise healthy individuals are well described (Isner et al., 1986; Karch and Billingham, 1988; Kloner et al., 1992; Kolodgie et al., 1995; Minor et al., 1991). A major target in which cocaine exerts its detrimental effects is the coronary vasculature. The recreational use of cocaine is thought to be associated with vasospasm of normal and atherosclerotic coronary arteries, coronary thrombosis, endothelial dysfunction, and increased accumulation of atherosclerotic plaque. However, limited information is available concerning the underlying pathophysiologic mechanism(s) of cocaine-induced vascular toxicity. Specifically, little is known of the effects of cocaine on the morphologic, biochemical, and functional properties of endothelial cells. Cocaine has been shown to disrupt the balance of endothelial prostacyclin and thromboxane production, which may be related to the increased tendency toward thrombosis and vasospasm observed in some cocaine abusers (Cejtin et al.,

Academic Press

1 The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or reflecting views of the Army, Navy, Air Force, or Department of Defense. 2 To whom correspondence and reprint requests should be addressed. Fax: (202) 782-9021. E-mail: [email protected].

0014-4800/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.

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110 1990; Eichhorn et al., 1992). It has also been suggested that cocaine may suppress endothelial cell growth (Welder et al., 1993), cause focal loss of endothelial cell integrity, or produce areas of extensive endothelial cell sloughing (Gilloteaux and Dalbec, 1991; Jones and Tackett, 1990). These effects may be critical to the development of accelerated atherosclerosis associated with cocaine abuse. The endothelium provides a selective barrier to macromolecules in the blood and injury to this barrier is associated with the pathogenesis of inflammatory and atherosclerotic diseases (DiCorleto and Gimbrone, 1996). Endothelial permeability is maintained principally by cytoskeletal elements that determine cell shape (Savion et al., 1982; Gotlieb, 1990), facilitate cell adherence to subcellular matrix (Wehland et al., 1979), and participate in the formation of junctional complexes (Wong and Gotlieb, 1986). The function of cytoskeletal elements is closely regulated by second messenger systems involving calcium, protein kinase C, and cyclic nucleotides (Hoek, 1992). The effects of cocaine on the endothelial cell cytoskeletal elements are poorly understood. At least in activated human platelets, acute exposure to cocaine causes a profound disruption in an otherwise well-organized cytoskeleton (Jennings et al., 1993). Whether direct exposure to cocaine alters the permeability function of vascular endothelial cells forms the basis of this investigation. In the present study, measurement of membrane permeability was performed in endothelial cell monolayers grown on porous filter supports. This model permits the direct assessment of both macromolecular transport and transmembrane resistance (Siflinger-Birnboim et al., 1987). Accordingly, experiments were designed to examine the effects of cocaine on the barrier function of human-derived umbilical vein endothelial cells. We also determined if the permeability changes induced by cocaine paralleled morphologic changes in endothelial cells.

MATERIALS AND METHODS Drugs and Solutions Stock solutions of cocaine hydrochloride (Sigma Chemicals, St. Louis, MO) and cocaine metabolites, benzoylecgonine, ecgonine methyl ester hydrochloride, and cocaethylene hydrochloride (Research Biochemicals International, Natick, MA) were prepared fresh in Dulbeccos phosphate-buffered saline (DPBS). Further dilutions were made in DPBS. Forskolin was purchased from Sigma and prepared as a stock

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solution in dimethylsulfoxide (DMSO); dilutions were made in DPBS. Endothelial Cell Isolation and Culture Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords collected postpartum by exposure to 0.2% dispase (Grade II, Boehringer Mannheim GmbH, W. Germany) for 18 h at 48C. Cells were initially seeded in 75-mm2 flasks coated with human fibronectin (Collaborative Biomedical, Bedford, MA) and grown to confluency (usually 3 to 4 days) in medium 199 (GibcoBRL, Grand Island, NY) with 10% fetal bovine serum (Hyclone, Logan UT), 10% human serum (Sigma) and endothelial cell growth factor. Cells were positively identified as endothelial by their cobblestone morphology, their reactivity to antibodies to human factor VIII (Sigma) as determined by indirect immunofluorescence, and their ability to take up acetylated lowdensity lipoprotein (LDL) (Stein and Stein, 1980). At confluency, cells were released with trypsin/EDTA and transferred to new dishes with a split ratio of 1:3 for further propagation. Endothelial cells used in the following study were passaged only once. Establishment of Monolayers for Passage Studies Polycarbonate micropore filters (Millicell-PCF, 12 mm diameter, 0.4 mm pore size, Millipore, Bedford, MA) were impregnated with 0.1% wt/vol gelatin (Difco, Detroit, MI) and 10 mg/ml human fibronectin (Collaborative). For passage and membrane resistance studies, endothelial cells were released by trypsin/EDTA and seeeded on filters at a density of 3 3 105 cells/cm2. Four to 6 h after seeding, the nonattached cells were removed. Filters were incubated at 378C in a humidified atmosphere of 95% air and 5% CO2 in culture medium as described above and endothelial cell monolayers were used for experimentation 48 h after seeding. Endothelial Permeability Prior to the experiment, endothelial cells were incubated for 1 h in minimum essential medium (assay media) supplemented with both 10% fetal bovine serum and 10% human serum. At the start of the experiment, either 5 mg/ml horseradish peroxidase (HRP type VI, Sigma) or 200 mg/ml of low-density lipoprotein labeled with the fluorescent molecule 1,18dioctadecyl-1-3,3,38,38-tetramethyl-indocarbocyanine perchlorate (DiI-labeled LDL; Organon Teknika Corp, Rockville, MD) in assay media was added in the presence or

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absence of varying concentrations of cocaine to the luminal chamber of the system. The fluid levels in the luminal and abluminal chambers were kept equal to avoid the effect of hydrostatic pressure across the monolayer. Experiments were performed at 378C in a humidified atmosphere containing 95% air and 5% CO2. Filters were kept on a gyrotory shaker during the course of the experiment to minimize unstirred layers. Transport experiments were performed in triplicate from a minimum of three different cell donors. The peroxidase concentration was determined spectrophotometrically as described previously (Langeler et al., 1989). The DiIlabeled LDL concentration was measured with use of a spectrofluorometer at an excitation of 553 nm and an emission of 578 nm (Hashida et al., 1986). The clearance of either peroxidase or DiI-LDL was used as an indicator of endothelial permeability (Silflinger-Birnboim et al., 1987). Clearance (ml) was calculated from: Cltracer 5 CAC 3 volumeAC (ml)/CLC, where CLC is the luminal tracer concentration at t 5 0, which did not change significantly during the course of the experiment; CAC is the abluminal tracer concentration; and volumeAC is the volume of media in the abluminal compartment. Clearance rate (ml/ min) at a certain point in time was determined by fitting the measured clearance for a single experiment (for each corresponding tracer) to a straight line by least-squares linear regression (StatView, Abacus Concepts, Inc., Berkeley, CA). The passage process for tracer molecules was consistently linear at least up to 4 h.

Endothelial Electrical Resistance The overall competency of the endothelial cell monolayers grown on filter supports was assessed by determining the impedance across the endothelium–filter barrier in a special chamber (Endohm; World Precision Instruments, Sarasota, FL). Transendothelial electrical resistance was determined by passage of a 20-mA AC square wave current at 50 Hz through the endothelial cell monolayer. The resulting resistance was measured by a volt–ohm meter (EVOM; World Precision Instruments). Endothelial cell resistance was calculated as the total resistance (filter, medium, and endothelial cells) minus the background resistance (filter and medium). The surface area of the filter insert was utilized to express the resistance values in ohms–cm2. To further confirm confluency, some endothelial monolayers cultured on filters were examined by scanning and transmission electron microscopy.

Endothelial Cytotoxicity Assay A 51Cr-release assay was used to assess the effects of cocaine on endothelial cell injury (Varani et al., 1992). Briefly, cells were seeded (7.5 3 104 cells/well) into wells of a 24-well dish in growth media. Twenty-four h later, monolayers were treated with 10 mCi/well Na251CrO4 (Amersham Corp., Arlington Heights, IL). After 18 h of labeling, the cells were washed twice with HBSS containing 10 mM HEPES to remove unincorporated 51Cr and then treated with medium 199 containing 10% FBS in the presence and absence of varying doses of cocaine for 6 h. The medium was collected and cells were extracted with 0.5% Triton X-100. Both medium and cell extract were counted; the percentage chromium release was calculated as [media counts/ (media 1 cell) counts] 3 100.

Endothelial Morphology The morphologic effects of cocaine exposure were assessed on confluent endothelial cell monolayers grown on glass coverslips. Endothelial cells were exposed to cocaine (1 mmol/L) with or without 5 min of pretreatment with forskolin (10 mM). The duration of treatment was 30 min prior to fixation and staining procedures. Control wells were treated with either PBS or 0.1% DMSO, respectively. Identification of cell borders with silver staining. Silver staining was used to identify cell borders to assess the morphology and orientation of endothelium. Endothelial cells with well-developed adherens and tight junctions stain with silver (Poole et al., 1958). The silver grains are thought to react with polyanionic structures present on these intercellular junctions (Zand et al., 1985). For the present studies, endothelial monolayers were stained with silver nitrate according to the methods of Smirnov et al. (1989). In brief, monolayers were washed in 5% sucrose, treated with 0.4% AgNO3 (Sigma) for 5 to 10 s, rinsed again with sucrose, and fixed with 4% paraformaldehyde. To intensify silver staining of endothelial cell borders, monolayers were subjected to ultraviolet light for several minutes. After staining, endothelial cell monolayers were visualized by phase-contrast or epifluorescence optics on a Nikon Diaphot microscope. Immunostaining of cell borders with PECAM-1. The 130- to 120-kDa integral membrane glycoprotein PECAM1 (platelet/endothelial cell adhesion molecule-1) is concentrated at endothelial intracellular junctions (Muller et al., 1989; Albelda et al., 1991), and immunocytochemical staining with PECAM-1 closely resembles silver staining (Smeets

112 et al., 1992). For immunodetection of PECAM-1, monolayers were washed twice with medium 199, fixed with methanol, air dried and incubated with a 1:10 dilution of PECAM-1 monoclonal antibody (Cal Tag, South San Francisco, CA). A goat anti-mouse Ig antibody labeled with rhodamine was used for visualization of bound PECAM-1. Localization of F-actin. Endothelial monolayers were stained with rhodamine phalloidin (Molecular Probes, Eugene, OR) which is specific for F-actin. The cells were briefly rinsed with PBS, fixed in 4.0% paraformaldehyde, and permeabilized with 0.5% Triton X-100 for 5 min. After washing with PBS, endothelial monolayers were incubated with 22 nmol/L rhodamine phalloidin in PBS for 20 min. The cells were then rinsed in PBS and mounted over glycerol/PBS 1:1).

Extraction and Analysis of cAMP At the start of the experiment, confluent endothelial cell monolayers, cultured in six-well plates, were rinsed twice with medium 199, and agents were added for the indicated time intervals. The reactions were terminated by washing the cells three times with ice-cold phosphate-buffered saline, and cAMP was extracted with 5% ice-cold trichloroacetic acid (TCA). The TCA precipitates were scraped from the dishes with a rubber policeman, transferred to micro-centrifuge tubes, and centrifuged at 12,000g for 10 min at 48C. The TCA was removed from the supernatant by repeated extraction (four times) with water-saturated ether, and the samples were dried under reduced pressure using a Speedvac concentrator (Savant Instruments Inc., Farmingdale, NY). Prior to analysis, the dried samples were reconsitituted in 0.4 ml sodium acetate (pH 6.2) (cAMP 3H Assay System; Amersham Corp.). Analysis of cAMP was conducted as described in the manufacturer’s direction for the assay. Recovery of cAMP was determined in preliminary experiments by use of radiolabeled nucleotide and liquid scintillation counting and was found to be greater than 75%.

Statistical Analysis The represented data are expressed as means 6 S.E. Differences between treatment groups were compared using a two-factor analysis of variance and Scheffe’s multiple-range test. For all analyses, statistical significance was established if the p value was #0.05.

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RESULTS Endothelial Cells as a Functional Barrier on Filter Supports To confirm that cellular junctions were established, the electrical resistance property of the endothelial cell monolayers was assessed prior to experimentation. The electrical resistance of confluent endothelial monolayers was 14 6 1 V?cm2 (20 determinations). This value is in agreement with previous studies using human umbilical vein-derived endothelial cells (Yamada et al., 1990). Scanning electron microscopy of selected endothelial cells grown on filter supports 48 h after seeding revealed a postconfluent culture showing the typical cobblestone architecture (data not shown). Transmission electron microscopy of endothelial cell monolayers showed several electron-dense junctional structures in focal areas of intercellular junctions (evidence of direct cell–cell contact) and occasional Weibel– Palade bodies. Effects of Cocaine on Endothelial Permeability In the absence of endothelial cells, both peroxidase and DiI–LDL passed freely through gelatin/fibronectin-coated polycarbonate membranes (data not shown). In filters seeded with endothelial cells, cocaine at a concentration of 0.01 mmol/L was associated with a slight increase in the permeability to peroxidase. This permeability change became increasingly significant at cocaine concentrations of 0.1 to 1 mmol/L (Fig. 1A). Cocaine (1 mmol/L) increased peroxidase clearance approximately five-fold from 0.04 6 0.008 to 0.20 6 0.034 mL/min. Further transport experiments examined the effect of cocaine on the passage of LDL (Fig. 1B). Addition of cocaine (1 mmol/L) to the media resulted in an approximate threefold increase in DiI–LDL clearance from 0.018 6 0.001 to 0.56 6 0.01 ml/min. Effects of Cocaine on Transendothelial Cell Electrical Resistance Addition of cocaine to the media at doses of (0.01 to 1 mmol/L) resulted in a concentration dependent decrease in transendothelial cell electrical resistance (Fig. 2). Maximal changes in resistance properrties were observed with cocaine concentrations of 1 mmol/L (control filters, 15 6 0.3 vs cocaine-treated filters, 4 6 0.2 V?cm2). The cocaine-induced decrease in electrical resistance was fully reversible following washout of the drug (Fig. 3).

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FIG. 2. Bar graph demonstating the change in transendothelial cell electrical resistance following cocaine treatment. Electrical resistance reported as V?cm2 was determined at 10 min incubation in untreated (Cont) and cocaine-treated monolayers. A concentration-dependent decrease in transendothelial membrane resistance was observed with a maximal change in resistance at 1 mmol/L cocaine. The values represent the mean 6 S.E. and experiments were performed in at least four different isolates. TEER, transendothelial electrical resistance. *Represents a significant increase (P # 0.05) in membrane resistance over control.

only a slight decrease in membrane resistance. In contrast, cocaine ethyl ester (1 mmol/L) resulted in a response virtually identical to that of the parent drug cocaine. FIG. 1. Bar graph demonstrating the effects of cocaine on the clearance (ml/min) of horseradish peroxidase (HRP; A) and low density lipoprotein (LDL; B) across endothelial monolayers. Clearance assays were performed on endothelial cells grown on gelatin/fibronectincoated filter supports as described under Materials and Methods. (A) A concentration-dependent increase in HRP clearance is seen with maximal clearance at 1 mmol/L cocaine. All values shown represent mean 6 S.E. and HRP experiments were performed in triplicate using six different isolates. (B) Cocaine at 1 mmol/L significantly increased the permeability of LDL labeled with the fluorescent indicator molecule 1,18dioctadecyl-1-3,3,38,38-tetramethyl-indocarbocyanine perchlorate (DiI). LDL experiments were performed in triplicate using four different isolates. *Represents a significant increase (P # 0.05) in HRP or LDL clearance over control.

The effects of the major metabolites of cocaine (benzoylecgonine, ecgonine methy ester, and cocaethylene) on electrical resistance were assessed (Fig. 4). Addition of 1 mmol/L of either benzoylecgonine or ecgonine methyl ester produced

Effects of Cocaine on Cell Viability Cocaine in concentrations up to 1 mmol/L did not cause an increase in 51Cr release compared with control cultures (percentage killed: control cells, 8.5 6 0.2 vs cocaine-treated cells, 8.1 6 0.5).

Neutralization of the Effect of Cocaine by Activation of Adenylate Cyclase Addition of forskolin (10 mM) to endothelial cell monolayers resulted in a significant (P 5 0.037) increase in electrical resistance 24.6 6 0.3 vs control 17.7 6 0.9 V?cm2 (Fig. 5). The marked decrease in electrical resistance in cocainetreated monolayers was significantly attenuated in cells pretreated with forskolin (Fig. 5).

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FIG. 3. Line graph showing the reversal of the effect of cocaine on transendothelial membrane resistance following washout of the drug. The electrical resistance across the endothelial cell monolayer rapidly decreased over time upon addition of 1 mmol/L cocaine and was promptly restored ('15 min) following washout (w/o) of the drug. The values represent the mean 6 S.E. of al least four independent experiments using different cell isolates.

Effect of Cocaine on Endothelial Morphology Representative light photomicrographs of confluent monolayers of endothelial cells cultured on gelatin/fibronectincoated coverslips and exposed to 1 mmol/L cocaine for 30

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min are shown in Fig. 6. Control endothelial monolayers demonstrated a normal cobblestone morphology as outlined by silver (Fig. 6A). However, argyrophilic junctions after cocaine treatment showed the formation of intercellular gaps (Fig. 6B). Cocaine-induced alterations in endothelial morphology were prevented by the addition of 10 mM forskolin (Fig. 6C). Endothelial cells with and without cocaine treatment were subsequently stained with a monclonal antibody directed against intergral membrane glycoprotein PECAM-1. Immunocytochemical staining with PECAM-1 demonstrated a cobblestone pattern closely resembling that of silver. Control monolayers showed intense staining of PECAM-1 at intercellular junctions (Fig. 6D). PECAM-1 staining of cocainetreated endothelial monolayers showed a focal decrease in the intensity of cell-to-cell staining and revealed gaps between cells (Fig. 6E). As with silver staining, forskolin prevented the formation of intercellular gaps induced by cocaine (Fig. 6F). The rhodamine phalloidin-stained actin filaments in control endothelial cells were arranged as linear stress fibers and dense peripheral bands (Fig. 6G). The dense peripheral bands of F-actin of adjacent cells were closely apposed to one another, and there was no intercellular gap formation. Cocaine treatment resulted in the loss of dense peripheral bands with an increase in centrally located stress fibers (Fig. 6H). In forskolin–cocaine-treated cells the endothelium became elongated, spindle-like, and showed marked thinning

FIG. 4. Bar graph demonstrating the effect of cocaine and its major metabolites benzoylecgonine, ecgonine methyl ester, and cocaethylene on transendothelial cell electrical resistance. All test agents were added at 1 mmol/L concentration and resistance properties were measured 10 min after stimulation. Cocaine metabolites benzoylecgonine and ecgonine methylester demonstrated a slight but nonsignificant decrease in electrical resistance (reported as as V?cm2), whereas the metabolite cocaethylene was equipotent to cocaine. The bars represent the mean 6 S.E. of three independent experiments using different cell isolates.

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FIG. 5. Bar graph demonstrating the inhibitory effect of the adenylate cyclase activator forskolin on the cocaine-induced decrease in transendothelial resistance. Endothelial cells were seeded on filter supports and treated with 1 mmol/L cocaine or a similar concentration of cocaine after 5 min pretreatment with 10 mM forskolin. Endothelial membrane resistance (reported as as V?cm2) was measured at 10 min after stimulation. Note, pretreatment with forskolin inhibited changes in membrane resistance produced by cocaine. The bars represent the mean 6 S.E. of four independent experiments using different cell isolates. *Represents a significant increase (P # 0.05) in membrane resistance over control.

of the dense peripheral band; no loss in the continuity of the monolayer was observed (Fig. 6I). Effect of Cocaine on the cAMP Concentration in Endothelial Monolayers Basal cAMP levels (2.99 6 0.49 pmol/9.6 cm2 of confluent cells) were similar at 5 (2.6 6 0.43 pmol/9.6 cm2) and 15 min (2.3 6 0.25 pmol/9.6 cm2) after treatment with 1 mmol/ L cocaine. In contrast, forskolin (10 mM) markedly increased endothelial cell cAMP (25.4 6 1.35/9.6 cm2) 15 min after exposure.

direct effects of cocaine on human endothelial cell function and the potential contribution of these effects to cocainemediated vascular toxicity. The present series of experiments demonstrates that acute cocaine exposure increases the permeability of cultured endothelial cell monolayers in the absence of cell detachment or lysis. Assessment of cell morphology in cocaine-treated endothelial monolayers showed a marked reorganization of F-actin and the formation of intercellular gaps. Cocaine-induced alterations in endothelial cell morphology and resistance were essentially neutralized by pretreatment with forskolin, however; cocaine had no effect on resting levels of intracellular cAMP. The cocaine concentrations used in our in vitro studies were comparable to plasma concentrations observed in cocaine abusers and cocaine-related deaths (plasma cocaine levels 6 3 1027 to 3 3 1024 M) (Poklis et al., 1985; Van Dyke et al., 1976). The so-called “binge users” of cocaine are known to use high doses for extended periods, oftentimes up to 200 h (Gawin, 1991). Plasma cocaine concentrations during a “binge” have not been documented although it is thought that the effects of cocaine on the cardiovascular system are especially significant during a binge. Cocaine-Accelerated Atherosclerosis Cocaine-induced injury of the vascular endothelium could be responsible for the development of accelerated atherosclerosis as observed in some habitual cocaine abusers (Dressler et al., 1990; Kolodgie et al., 1991, 1992; Mittleman and Wetli, 1987). Although our data show an increase in the transport of LDL in endothelial monolayers treated with cocaine, further experiments are required to show the conversion into an atherosclerotic plaque. We have previously demonstrated the atherogenic potential of cocaine in a rabbit model of diet-induced hypercholesterolemia (Kolodgie et al., 1993). While cocaine may enhance the permeability function of the vascular endothelium, thus allowing greater amounts of atherogenic lipoproteins to diffuse into the intima, this mechanism is merely suggestive, and conclusive evidence as to whether this occurs in vivo is still required. Cocaine and Lethal Cell Injury

DISCUSSION

The deleterious effects of cocaine on the cardiovascular system are well described. Much less understood are the

Acute exposure to cocaine in vitro does not appear to directly cause lethal toxicity to the endothelium. In contrast, a loss of endothelial cell integrity and excessive cell sloughing was observed by scanning electron microscopy in the femoral arteries from dogs after long-term exposure to 1 mg/kg intravenous cocaine for 4 weeks (Jones and

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FIG. 6. Photomicrographs of confl uent endothelial cells seeded on gelatin-coated glass coverslips and challenged with 1 mmol/L cocaine or a similar concentration of cocaine following 5 min of pretreatment with 10 mM forskolin (for details see Materials and Methods). Endothelial junctions were identifi ed by silver nitrate staining (A through C, magnifi cation3250) and antibodies to PECAM-1 (D through F, magnifi cation 3600). F-actin was observed following staining with rhodamine-phalloidin (G through I, magnifi cation3600). Arrows indicate interendothelial gaps. Note, cocaine treatment resulted in the formation of intercellular gaps and F-actin rearangement which was prevented by pretreatment with forskolin. Results are representative of three separate experiments on different isolates.

COCAINE INCREASES ENDOTHELIAL CELL PERMEABILITY

FIG. 6. Continued

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FIG. 6. Continued

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Tackett, 1990). In cultured rat heart-derived endothelial cells, cytotoxicity as measured by lactate dehydrogenase release was significantly elevated only after 24-h exposure to high doses of cocaine (1 3 103 M) (Welder et al., 1993). However, in the same report, lysosomal integrity and cell viability as determined by neutral red retention failed to demonstrate cocaine-induced cell injury at any of the doses studied. In the present series of experiments, changes in endothelial cell membrane resistance following acute exposure to cocaine (0 to 30 min) occurred in the absence of lethal endothelial cell injury as washout of the drug totally reversed its effect. The absence of cocaine-induced lethal toxicity was confirmed in a 6-h 51Cr-release assay. Morphologic studies demonstrated no evidence of cell detachment and/or lysis in endothelial cells treated with cocaine. Thus, data from our laboratory and others suggest that short-term exposure to cocaine in endothelial cells is relatively well tolerated. Perhaps, repeated cocaine exposure, cocaine metabolites (Schindler et al., 1995), or cocaine-associated adrenergic toxicity (Pettersson et al., 1990) may be responsible for the endothelial injury observed in vivo. Cocaine and the Endothelial Cytoskeletal Network Since the effect of cocaine on endothelial monolayers was reversible, we speculated that the underlying mechanism may involve alterations in the endothelial cytoskeleton. Endothelial cell are known to possess contractile activity closely regulated by cytoskeletal elements and second messenger systems. Early in vivo experiments showed that histamine produces a H1-receptor-dependent contraction of rat and rabbit venule endothelial cells (Majno et al., 1967). Direct microfilament disruption in endothelial cell monolayers with cytochalasin B results in increased permeability to albumin (Shasby et al., 1982). Evidence of increased paracellular macromolecular transport through interendothelial gaps has been demonstrated in endothelial cells from different origins treated with various cytokines (Koga et al., 1995; Maruo et al., 1992), biogenic amines (Langeler et al., 1989), thrombin (Garcia et al., 1986), or oxidative stress (Siflinger-Birnboim et al., 1992; Philips et al., 1988). The relative contribution of cocaine on endothelial cytoskeletal function prior to this study had not been tested, although cocaine treatment was associated with the disruption of cytoskeletal elements in activated platelets (Jennings et al., 1993). In the present study, cocaine treatment caused a rearrangement of F-actin (decreased dense peripheral bands with increased central stress fibers). The change in F-actin arrangement in cocaine-treated endothelial monolayers resulted in the formation of intercellular “gaps.” We believe

that the changes in endothelial cell shape induced by cocaine may be responsible for the increase in peroxidase and LDL permeability and the associated alterations in membrane resistance. Cocaine and Intracellular Signal Transduction A number of signal transduction pathways involved in the regulation of endothelial permeability and maintenance of endothelial integrity have been identified. The endothelial cytoskeletal network has been shown to be responsive to changes in cyclic nucleotides, protein kinase C activation, and calcium (Hoek, 1992). The complexities of how these various pathways interact are not well known. In the present study, induction of cAMP by forskolin increased transendothelial cell electrical resistance and resulted in marked changes in endothelial cell morphology. Similar findings have been reported in other laboratories using human umbilical vein-derived endothelial cells (Antonov et al., 1986; Yamada et al., 1990; Langeler and van Hinsbergh, 1991). The major function of cAMP within cells is allosteric activation of cAMP-dependent protein kinases that can phosphorylate a variety of cellular targets, including cytoskeletal proteins (Vallee et al., 1981). The phosporylation of cytoskeleton-associated proteins is thought to play a role in cytoskeleton assembly, which is a critical factor involved in the maintenance of cell–cell contact (Hoek, 1992). Interestingly, forskolin prevented the cocaine-induced decrease in transendothelial cell electrical resistance and formation of intercellular gaps. Inflammatory cytokines, such as tumor necrosis factor, have been shown to disrupt the barrier function of endothelial cells by reducing levels of cAMP through increased phosphodiesterase activity (Koga et al., 1995). However, in the present study such a mechanism does not appear to be evident since cocaine had no effect on basal concentrations of cAMP. Recent evidence suggests that intracellular levels of cAMP may affect actomyosin-initiated contraction of the endothelial cytoskeleton through the 20-kDa myosin light chain (MLC20) protein. Phosphorylation of MLC20 is thought to result in actomyosin contraction and centripetal tension (Schnittler et al., 1990; Wysolmerski et al., 1990). Increasing intracellular cAMP by forskolin or cAMP analogs decreases MLC20 phosphorylation in human umbilical vein-derived endothelium (Moy et al., 1993). In contrast, histamine, an agent known to disrupt endothelial cell permeability, increases MLC20 phosphorylation (Moy et al., 1993). Similar to our results of cocaine study, resting levels of intracellular cAMP are unchanged by histamine, whereas forskolin or cAMP analogs prevent the disruption of endothelial cell

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permeability induced by this agent (Carson et al., 1989). The preservation of endothelial cell barrier function by cAMPelevating agents is thought to be afforded by their ability to suppress phosphorylation of MLC20 induced by histamine (Moy et al., 1993). These data suggest that MCL20 may be an important regulatory protein in the control of endothelial barrier function; whether cocaine exerts its permeability effects through a similar mechanism remains to be determined. Moreover, MLCK-independent pathways have also been implicated in disruption of endothelial cell contact by inhibition of protein kinase A and tyrosine kinase activities, in particular, the p125 focal adhesion kinase (Garcia et al., 1997). Alternatively, cocaine may effect endothelial cell membrane permeability by interfering with normal calcium homeostasis. Certain anesthetics cause displacement of 45Ca2+ binding to the cytoplasmic surface of plasma membranes (Low et al., 1978); it is possible that changes in permeability by cocaine may occur by evoking the release of cytoplasmic calcium from intracellular stores. In rat-heart-derived endothelium, loaded with the fluorescent Ca2+-indicator dye fura2, acute cocaine exposure in concentrations similar to those of the present study increased levels of intracellular calcium (Welder et al., 1993). Therefore, cocaine-induced disturbances in intracellular calcium may be responsible for the permeability changes observed in human endothelial cells in this study.

In humans, cocaine is rapidly metabolized, and less than 1% is excreted in the urine (Ambre, 1985; Jeffcoat et al., 1989). The drug’s principal metabolites are benzoylecgonine and ecgonine methyl ester, in addition to other numerous degradation products formed in smaller amounts (Zhang et al., 1990). None of the principal metabolites are active psychomotor stimulants; however, in some instances they do posses physiologic activity. For example, in feline cerebral arteries, benzoylecgonine is a potent vasoconstrictor, whereas ecgonine methyl ester causes mild vasorelaxation (Madden and Powers, 1990). A novel psychotropic metabolite of cocaine, cocaethylene, has been identified in individuals abusing cocaine and alcohol concurrently (Hearn et al., 1991; Jatlow et al., 1991). The longer half-life of cocaethylene (2 h vs about 38 min for cocaine) may result in a greater potential for cardiotoxicity than its parent drug (Hern et al., 1991; Randal, 1992), but little is known of the physiologic activity of this metabolite (Rose, 1994). In the present study, benzoylecgonine and ecgonine methyl ester had little or no effect on membrane resistance, whereas cocaethylene was found to be equipotent to cocaine. This finding may have important clinical implications because plasma concentrations of cocaethylene in man have been reported to exceed that of cocaine itself (Hern et al., 1991).

Cocaine/Endothelium/ and Nitric Oxide Synthase

Study Limitations

Interactions among intracellular levels of calcium, cAMP, and cGMP most likely regulate cytoskeletal organization and endothelial permeability. One system that generates cGMP (and is influenced by calcium) is constitutive nitric oxide synthase (NOS), which produces nitric oxide or NO. Recent studies indicate that NO modulates vascular permeability under both physiological and pathophysiological conditions (Draijer et al., 1995). Since NOS inhibitors have been shown to influence cocaine-induced toxicity (Itzhak et al., 1993; Pudiak and Bozarth, 1993; Brogan, 1991), we hypothesized that cocaine-induced increase in permeability may be affected by NO. However, unlike other endothelial cells, NO production in HUVECs is limited by the decline in tetrahydrobiopterin that occurs in culture (RosenkranzWeiss et al., 1994). Although HUVECs in our study were subcultured only once, we were unable to detect NO production by bioassay in control or cocaine-treated cells, perhaps resulting from a loss in essential cofactors for NOS (unpublished observation). At least in our model, it appears that decreased NO production is unlikely to be responsible for the permeability effects of cocaine.

Cocaine Metabolites and Endothelial Cell Permeability

Because the permeability characteristics of endothelial cells differ among species and vascular sites (Hoek, 1992), the findings in the present investigation are applicable only to human umbilical vein endothelial cells and not necessarily to systemic arteries. Also, the movement of solutes across endothelial cell membranes can occur by a transcellular mechanism involving endocytosis. Whether cocaine has an effect on the transcytosis of macromolecules is unknown.

SUMMARY

Our study supports a role of cocaine-mediated changes in endothelial cell permeability as a potential underlying mechanism of cocaine-induced vascular toxicity. Whether permeability changes induced by cocaine demonstrated in vitro explains the accelerated atherosclerosis observed in some cocaine abusers remains to be determined. The toxicity

COCAINE INCREASES ENDOTHELIAL CELL PERMEABILITY

of cocaine metabolites, in particular cocaethylene, on endothelial cell membrane permeability may have importance in individuals who abuse cocaine and alcohol concurrently.

ACKNOWLEDGMENTS

This study was supported, in part, from a grant (No. 2284) provided by the American Registry of Pathology.

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