It has been estimated that >23 million people in the United States have used cocaine at some time. The epidemic increase in cocaine abuse during the last decade has focused public and scientific attention on the cardiovascular consequences of unrestricted use of the drug. Experimental data have shown that cocaine administration depresses myocardial contractility, increases blood pressure, and induces coronary artery vasoconstriction and myocardial ischemia (1-4).
The effect of cocaine on the cardiovascular system is multifactorial. Whereas the cardiovascular effects of cocaine are often attributed to its potent pharmacologic effects as an indirect-acting sympathomimetic agent, direct cytotoxic effects of cocaine on the cardiovascular system remain unclear. Recently, several animal experiments have reported that cocaine can induce apoptosis in cultured myocytes (5), neurons (6), thymocytes (7), and hepatocytes (8). Apoptosis is an active physiologic process that permits the removal of unwanted or damaged cells from the body through an intrinsic cell-suicide program. Compelling evidence has indicated that apoptotic cell death also may play a critical role in a variety of cardiovascular diseases, including myocardial ischemia and infarction, heart failure, and atherosclerosis (9, 10).
We have recently demonstrated that cocaine causes a direct cytotoxic effect on fetal rat heart and induces apoptosis in cultured fetal rat myocardial cells in a time- and dose-dependent manner (5). Cocaine-induced apoptosis in fetal myocytes was characterized by multiple morphologic and biochemical features of typical apoptotic cell death. The defining features of apoptotic cell death are characterized by morphologic changes such as membrane remodeling, DNA fragmentation, DNA condensation, cell shrinkage, and formation of apoptotic bodies. Cocaine-induced apoptosis also was demonstrated in adult rat heart (11). Repeated intraperitoneal and intravenous cocaine exposure in the rat has been found to increase apoptotic cell death in the heart from 2% in control rats to 14% in cocaine-treated rats. Contraction-band necrosis was not found in either group.
Little is known about the cytotoxic effects of cocaine on the endothelium. This study was designed to test the hypothesis that cocaine causes endothelial cell injury by inducing apoptosis in cultured human coronary artery endothelial cells (HCAECs). Cocaine-induced apoptosis was determined by examining apoptotic features of cell and nucleus morphology and DNA fragmentation. By using cyclosporin A to block cytochrome c release from the mitochondria, we examined the potential role for the mitochondria in cocaine-mediated apoptosis in HCAECs. The involvement of mitochondrial pathway was further examined by using the caspase-9 inhibitor. We also examined the potential role of opioid receptors and intracellular calcium in cocaine-induced apoptosis in the coronary artery endothelial cells.
HCAECs, endothelial cell growth medium with 5% fetal bovine serum (EGM-MV Bulletkit), and trypsin reagentpack were purchased from Clonetics Co. (Walkersville, MD, U.S.A.). Cocaine, nifedipine, naloxone, cyclosporin A, Annexin V kit, Streptavidin-Fluorescein Isothiocyanate (Sav-FITC), Hoechst 33258, methyl green, and phosphate-buffered saline (PBS) were purchased from Sigma (St. Louis, MO, U.S.A.). Ac-DEVD-CHO, caspase-3 inhibitor, was from Pharmingen (San Diego, CA, U.S.A.). Z-LEHD-FMK, caspase-9 inhibitor, was from Kamiya Biomedical Company (Seattle, WA, U.S.A.). Proteinase K, in situ cell death detection kit, DNase-free RNase, and DNase were from Boehringer Mannheim (Indianapolis, IN, U.S.A.). SYBO Gold was from Molecular Probes (Eugene, OR, U.S.A.).
Third-passage HCAECs were seeded at a density of 5,000/cm2 and cultured in EGM-MV Bulletkit, which contains endothelial cell basal medium, 500 ml; 10 μg/ml hEGF, 0.5 ml; 50 mg/ml gentamicin and 50 μg/ml amphotericin-B, 0.5 ml; 1 mg/ml hydrocortisone, 0.5 ml; 3 mg/ml bovine brain extract, 2 ml; and fetal bovine serum (FBS), 25 ml. The cells were cultured at 37°C in a humidified incubator with 5% CO2, 95% air, and were used for the experiments at the fifth and sixth passages of 80% confluence. Twenty-four hours before cocaine treatment, the medium was replaced with the serum-free medium. Cells were then exposed to different doses of cocaine (10-500 μM) for various periods. The morphologic changes of endothelial cells were examined by phase-contrast and fluorescence microscopy.
Detection of cell-surface phosphatidylserine
Cell-surface phosphatidylserine was detected by phosphatidylserine-binding protein annexin V conjugated with streptavidin-fluorescein isothiocyanate (FITC) by using the commercially available Annexin V kit (Sigma). Monolayers of HCAECs grown on coverslips were washed with cold PBS, and incubated with 100 μl annexin V incubation reagent (10 × binding buffer, 10 μl; propidium iodide, 0.5 μg/10 μl; annexin V conjugate, 0.05 μg/l μl; distilled water, 79 μl) per sample for 15 min at room temperature in the dark. Cells were then washed with 1 × binding buffer and incubated with 100 μl of 1 × binding buffer containing FITC for 15 min at room temperature in the dark. After washing with 1 × binding buffer, the sample was examined immediately by fluorescence microscopy. The combination of propidium iodide and annexin V conjugated with FITC in the kit allowed the differentiation between the early (annexin V-FITC positive) and late (annexin V-FITC and propidium iodide positive) apoptotic cells, and viable cells (unstained).
DNA fragmentation on agarose gel
After treatments, cells were harvested and lysed in a lysis buffer of 20 mM Tris (pH 8.0), 20 mM EDTA, and 1% SDS containing 300 μg proteinase K at 55°C for 60 min. Protein was removed by the addition of sodium acetic acid and centrifugation. DNA was precipitated from the supernatant with the same volume of isopropanol and centrifuged at 14,000 g for 1 min. The pellet was washed by 70% ethanol twice at 4°C. DNA was dissolved in Tris-EDTA buffer containing 0.5 U DNase-free RNase A and incubated for 2 h at 37°C. DNA (10 μg) was electrophoresed at 70 V in a 1.8% agarose gel in Tris-phosphate-EDTA buffer, stained with SYBO Gold, and photographed with UV illumination. A 100-bp DNA ladder molecular-weight marker was added to each gel as a reference for analysis of internucleosomal DNA fragmentation.
In situ TUNEL assay
In situ labeling of fragmented DNA was performed with terminal deoxynucleotidyl transferase (Tdt) UTP nick end-labeling (TUNEL) with a commercially available in situ cell death detection kit (Boehringer Mannheim) according to the manufacturer's instruction. In brief, monolayers of HCAECs grown on coverslips were fixed with 4% paraformaldehyde solution for 30 min at room temperature, washed with PBS, and incubated with permeabilization solution (0.1% Triton X-100, 0.01% sodium citrate) for 7 min at room temperature. Apoptotic cells were labeled with 50 μl TUNEL reaction mixture, and conjugated with alkaline phosphatase (AP) by incubating them with 50 μl converter-AP for 30 min at 37°C. After substrate reaction, stained cells were analyzed under light microscope. The nuclei were counterstained with 0.5% methyl green for 2 min at room temperature. Negative control samples for TUNEL staining lacked Tdt. Positive controls were performed by incubating fixed and permeabilized cells with DNase 1U/100μl mixture for 10 min at room temperature.
Quantitative analysis of apoptotic cells
Fluorescent DNA-binding dyes were commonly used to define nuclear chromatin morphology as a quantitative index of apoptosis within a cell culture system (12,13). Cells grown on coverslips were washed with PBS and then fixed in methanol/acetic acid (3:1) at 4°C for 5 min. After fixation, the cells were stained for 10 min with the fluorescent DNA-binding dye Hoechst 33258 at 8 μg/ml, and nuclear morphology was examined by fluorescence microscopy. Individual nuclei were visualized at ×400 to distinguish the normal uniform nuclear pattern from the characteristic condensed coalesced chromatin pattern of apoptotic cells. To quantify apoptosis, 500 nuclei from random microscopic fields were analyzed, and the percentage of apoptotic cells was calculated as the number of apoptotic cells/number of total cells × 100%. Each experiment was conducted in triplicate and repeated 3 to 4 times.
Data are presented as the mean ± SEM. Statistical analysis was performed with one-way analysis of variance (ANOVA) followed by Newman-Keuls tests. Values were considered statistically significant at p < 0.05.
Effect of cocaine on apoptotic cell death in HCAECs
Figure 1 shows cocaine-induced phosphatidylserine translocation from the inner to the outer leaflet of the plasma membrane detected by the phosphatidylserine-binding protein annexin V conjugated with FITC. Without membrane permeabilization, no staining of control HCAECs was observed. After treatment of the cells with cocaine (100 μM) for 6 h, binding of annexin V-FITC to the surface of apoptotic HCAECs was observed (Fig. 1A). No staining of the apoptotic HCAECs with trypan blue was detected at this stage. In addition, propidium iodide failed to stain apoptotic HCAECs at 6 h treatment of cocaine (Fig. 1A). After 24-h treatment, propidium iodide penetrated the apoptotic cells and stained nuclear DNA, producing a strong, yellow-red fluorescent signal (Fig. 1B).
Fig. 2 shows nuclear chromatin morphology by Hoechst 33258 staining using fluorescence microscopy and indicates condensed, coalesced, and segmented nuclei induced by cocaine. Cocaine-induced DNA fragmentation in HCAECs was also detected in situ by TUNEL assay. As shown in Fig. 3, cocaine (100 μM for 24 h) increased the apoptotic nuclei identified by terminal deoxy transferase labeling of 3′ DNA ends. In accordance, cocaine induced formation of oligonucleo-some-sized fragments of DNA as ladders of ∼200 bp on agarose gels in a time-dependent manner (Fig. 4). Fig. 5 shows representative cell morphological changes induced by cocaine. Under control conditions, HCAECs morphology appears normal and cell density is approaching confluence (Fig. 5A). After exposure to cocaine (100 μM for 24 h), the cells exhibited the characteristic features of cell shrinkage, rounding and partial detachment, and demonstrated the lobulated appearance of apoptotic cells (Fig. 5B).
Quantification of cocaine-induced apoptotic nuclei defined by the fluorescent DNA-binding dye Hoechst 33258 indicated that cocaine induced apoptosis in HCAECs in a time- and dose-dependent manner. Figure 6 shows that cocaine time-dependently induced apoptosis in HCAECs. At 24 h of incubation, cocaine (100-500 μM) produced concentration-dependent increases in apoptotic cells in HCAECs (Fig. 7, bottom). Prolonged treatment (72 h) lowered the effective dose of cocaine to 10 μM (Fig. 7, top). The control level of apoptotic cells also was increased from 8.1 ± 0.6% to 26.1 ± 1.2%.
Effect of cyclosporin A and caspase inhibitors on cocaine-induced apoptosis
As shown in Fig. 8,, cyclosporin A inhibited cocaine-induced apoptosis in HCAECs in a dose-dependent manner with a pD2 of 6.54 ± 0.18. The maximal inhibition of 62% was obtained at 3 μM cyclosporin A. However, higher dose of cyclosporin A (10 μM) itself caused a significant increase of apoptotic cells compared with the control group (data not shown), suggesting a toxic effect of cyclosporin A on HCAECs at higher doses. Both caspase-3 (Ac-DEVD-CHO) and caspase-9 (Z-LEHD-FMK) inhibitors blocked cocaine-induced apoptosis in HCAECs (Fig. 9).
Effect of nifedipine and naloxone on cocaine-induced apoptosis
To elucidate the role of calcium in cocaine-induced apoptosis, HCAECs were incubated concomitantly with cocaine and various concentrations of nifedipine for 24 h. Figure 10 shows that nifedipine (0.1-10 μM) inhibited cocaine-induced apoptosis in a dose-dependent manner (p < 0.05). Nifedipine (10 μM) did not have an apoptotic effect on HCAECs but completely blocked the cocaine-induced apoptosis (Fig. 10). In addition, naloxone dose-dependently inhibited cocaine-induced apoptosis (Fig. 11; p < 0.05), suggesting an involvement of opioid receptors in the cocaine-induced apoptosis in HCAECs.
This study demonstrated for the first time that cocaine causes a direct cytotoxic effect on cultured HCAECs and induces apoptosis in HCAECs in a time- and dose-dependent manner. Our data suggest that cocaine-induced apoptosis in HCAECs is mediated by opioid receptors and is calcium dependent. In addition, the release of cytochrome c and its subsequent activation of caspase-9 and caspase-3 are likely to play a key role in cocaine-induced apoptosis in HCAECs.
The finding of annexin V-FITC binding to HCAECs after 6-h treatment of the cells with cocaine (Fig. 1A) suggests that cocaine induces apoptosis in HCAECs as early as at 6 h. In normal cells, the restriction of membrane lipid phosphatidylserine to the inner leaflet of the plasma membrane precludes the binding of phosphatidylserine-binding proteins to healthy cells. Phosphatidylserine externalization occurs rapidly after apoptosis and precedes other morphologic changes such as "leaky" plasma membrane, nuclear breakdown, and chromosomal fragmentation. As a consequence, the phosphatidylserine-binding protein annexin V is able to bind to the apoptotic cells. It has been demonstrated that annexin V-FITC binding is a specific and sensitive method to identify the early stage of apoptosis (14-16). As reported in many other cells (14-16), annexin V binding preceded the loss of membrane integrity in apoptotic cells, induced by cocaine in our study. This conclusion is supported by the finding that propidium iodide failed to stain apoptotic HCAECs at 6 h (Fig. 1A). In addition, no staining of the apoptotic HCAECs with trypan blue was detected at this stage. Because phosphatidylserine is a potent surface procoagulant, a recent study demonstrated that human endothelial cells with phosphatidylserine externalization during apoptosis were markedly procoagulant (14). This procoagulant effect was entirely abolished by annexin V, confirming that it was phosphatidylserine dependent. This finding suggests that cocaine-induced phosphatidylserine externalization in HCAECs may cause pathologic intravascular coagulation events and impair coronary circulation, which may explain in part cocaine-induced myocardial ischemia and infarction. The finding of propidium iodide staining after 24-h incubation of cocaine (Fig. 1B) suggests that the plasma membrane becomes increasingly permeable during the later stages of apoptosis in HCAECs.
In this study, cocaine-induced apoptosis in HCAECs also was clearly demonstrated by morphologic changes such as cell shrinkage and rounding, characteristic features of apoptotic death. Moreover, simultaneous assessment of nuclear chromatin morphology verified that these cells eventually manifested typical apoptotic condensed and fragmented nuclei. In addition, we have confirmed that the process of apoptosis, defined on the basis of cellular and nuclear chromatin morphology, correlates with apoptosis, defined on the basis of internucleosomal DNA fragmentation assessed by in situ labeling and gel electrophoresis. Similar findings of cocaine-induced apoptosis have been reported in fetal rat myocytes (5), mice hepatocytes (8), and fetal mice cortical neurons (6). Apoptosis in endothelial cells has been demonstrated with various factors such as oxidized low-density lipoprotein (13,17,18), adenosine triphosphate (ATP) and adenosine (19), lipopolysaccharide (12), and glucose (20). Our finding of the maximum of 23% apoptotic cells induced by cocaine is in agreement with the previous studies showing that 18-21% of human umbilical vein and coronary artery endothelial cells, respectively, became apoptotic in the presence of the maximal dose of oxidized low-density lipoprotein (13,18). Because serum levels of cocaine in active drug abusers are often >100 μM(6), the finding that cocaine can induce apoptosis in HCAECs at concentrations as low as 10 μM in our study fully warrants its pathophysiological relevance. Whereas the half-life of cocaine in the serum ranges from 1 h (intravenous dosing) to 5 h (nasal dosing), the repeated uses of cocaine in active drug abusers produces dose-related increases in serum cocaine concentrations (21,22).
The mechanisms underlying cocaine-induced apoptosis in HCAECs are not clear at present. It has been demonstrated that cocaine activates immediate-early genes such as c-fos, c-jun, and zif/268(23), which have been implicated in certain forms of apoptosis (9). Our finding that cocaine-induced apoptosis was blocked by nifedipine suggests that calcium influx plays a key role in cocaine-induced apoptosis in HCAECs. Similar findings were observed in human umbilical vein endothelial cells in which oxidized low-density lipoprotein-induced apoptosis was mediated by calcium influx (17). Whereas early evidence suggested that the endogenous endonuclease implicated in apoptosis in most model systems is calcium dependent (24-26), recent studies have demonstrated that molecular targets for calcium include signal-transduction intermediates, endonuclease and proteases, and the enzymes involved in the maintenance of phospholipid asymmetry in the plasma membrane (27). Moreover, it has been shown that calcium inhibits Bcl-2 protein activity (28), induces a switch from low- to high-conductance state of mitochondria permeability transition pore (29), and activates caspase-3 activity (30).
In many, if not all, apoptosis scenarios, there is an opening of the mitochondrial permeability transition pore leading to collapse of the mitochondria inner transmembrane potential and the release of cytochrome c (31). It has been demonstrated that cocaine increases intracellular calcium (32-34) and impairs mitochondrial function by dissipating mitochondria membrane potential (32,35,36). Our finding that cyclosporin A dose dependently inhibited cocaine-induced apoptosis in HCAECs suggests that release of cytochrome c from the mitochondria plays a key role in cocaine-induced apoptosis in HCAECs. It has been well documented that cyclosporin A prevents cytochrome c release by stabilizing the mitochondrial transmembrane potential, and inhibits apoptosis (31,37-39). In this study, we have demonstrated that cyclosporin A inhibits cocaine-induced apoptosis in HCAECs in a dose-dependent manner with an IC50 of 0.3 μM. This finding is consistent with our previous study in which we have demonstrated that cocaine-induced cytochrome c release and apoptosis in myocytes are inhibited by cyclosporin A (5). Similar findings also were obtained in human endothelial cells in which cyclosporin A dose dependently inhibited oxLDL-induced cytochrome c release and apoptosis (39). In contrast to the previous finding (39), present studies demonstrated that a high concentration of cyclosporin A (10 μM) itself induced apoptosis in HCAECs. Similar findings were demonstrated in other cells (40,41). The genotoxic properties of cyclosporine have been documented (42-44). The possibility that cyclosporin A inhibits the cocaine-induced apoptosis by its interference at the level of the cell cycle remains elusive.
The notion that cocaine-induced apoptosis in HCAECs may involve release of cytochrome c from the mitochondria has been further supported by the studies of caspase inhibitors on cocaine-induced apoptosis. It has been well documented that the caspase cascade includes both initiator caspases and effector caspases (31,45,46). Proapoptotic signals activate an initiator caspase that, in turn, activates effector caspases such as caspase-3, leading to apoptotic cell death. Two initiator caspases, caspase-8 and caspase-9, mediate distinct sets of death signals. Caspase-8 is associated with apoptosis involving death receptors that are activated by ligands of the tumor necrosis factor gene superfamily (45). In contrast, caspase-9 is involved in death induced by cytotoxic agents, and is activated by cytochrome c and Apaf-1 (31,46). In this study, we demonstrated that cocaine-induced apoptosis in HCAECs is blocked by caspase-9 and caspase-3 inhibitors, respectively. This finding reinforces the notion that cocaine-induced apoptosis in HCAECs is mediated by the mitochondrial pathway.
In summary, we have shown that cocaine induces a time- and dose-dependent increase in apoptosis in cultured HCAECs. Cocaine-induced apoptosis in HCAECs is calcium dependent and is likely to be mediated by the release of cytochrome c and subsequent activation of caspase-9 and caspase-3. Increased apoptosis of endothelial cells in the coronary artery results in endothelial dysfunction, which is likely to play a key role in cocaine-induced coronary artery vasoconstriction, leading to myocardial ischemia and infarction. In addition, apoptosis has been proposed as a means of presenting otherwise sequestered antigens to the immune system (47,48). As a consequence, autoantibodies could bind to apoptotic cells and trigger an inflammatory response that damages surrounding healthy tissues.
Acknowledgment: This work was supported in part by NIH grants HL-54094, HL-57787, a Grant-in-Aid from the American Heart Association #96007560, and by Loma Linda University School of Medicine.
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