Cocaine Induces Apoptosis in Primary Cultured Rat Aortic Vascular Smooth Muscle Cells: Possible Relationship to Aortic Dissection,Atherosclerosis,and Hypertension

Abstract

Cocaine abuse is known to induce many adverse cardiovascular effects, including hypertension, atherosclerosis, and aortic dissection. A major physiological event leading to these pathophysiological actions of cocaine could be apoptosis. This study was designed to investigate if primary cultured rat aortic vascular smooth muscle cells (VSMCs) can undergo apoptosis when treated with cocaine. After treatment with cocaine (10⁻⁶ to 10⁻⁴ M), morphological analysis of aortic VSMCs using confocal fluoresence microscopy showed that the percentage of apoptotic aortic VSMCs increased after cocaine (10⁻⁶ to 10⁻⁴ M) treatment for 12, 24, and 48 h. These results demonstrate that aortic VSMCs can undergo rapid apoptosis in response to cocaine in a concentration-dependent manner. Cocaine-induced apoptosis may thus play a major role in cocaine abuse-induced aortic dissection, atherosclerosis, and hypertension.

Over the past decade, cocaine abuse has increased dramatically and has attracted much public and scientific attention into both the acute and chronic toxic effects of this drug of abuse. It is estimated that 25 million Americans have used cocaine, and 1.5 million use it on a regular basis. Among the many adverse effects, cardiovascular complications seem to very prominent, especially compared with other controlled drugs (Gold 1993). Long-term ingestion of cocaine is well known to induce hypertension (Brecklin and Bauman 1999) and accelerate atherosclerosis of coronary, cerebral, and peripheral arteries (Kolodgie et al. 1991; Di Paolo et al. 1997), which may contribute to complications of the heart and brain. Acute abuse of cocaine can induce aortic dissection and several cases of cocaine-induced aortic dissection have been reported since 1987 (Edwards and Rubin 1987; Hsue et al., 2002). Although aortic dissection is a complication that rarely happens, it is very dangerous and is an immediate threat to the lives of cocaine abusers once it happens, so it is of great interest to both physicians and medical scientists. Suggested mechanisms of cocaine-induced hypertension, atherosclerosis, and aortic dissection include vasospasm, depletion of intracellular free magnesium ions, increased calcium uptake, and increased plasma catecholamine concentration resulting from decreased re-uptake of catecholamines at the neural synapses caused by cocaine (Altura and Gupta, 1992; Altura et al. 1993; He et al. 1993; Sofuoglu et al. 2001). But the mechanisms at the cellular and molecular levels still remain to be elucidated.

Apoptosis (also known as programmed cell death) was first identified and so-named by Kerr in 1972 (Kerr, Wyllie, and Currie 1972). It is classically characterized by chromatin condensation and margination, cell shrinkage, and membrane blebbing. It is a kind of cell suicide quite different from necrosis, without any inflammatory cell infiltration. Apoptosis is well programmed by an intrinsic cell-suicide machinery, which involves activation of a special group of enzymes called caspases, cutting normal DNA molecules into small fragments, and disintegration of the cell into apoptotic bodies (for recent review, see Zimmermann, Bonzon, and Green 2001).

Apoptosis is not only an important physiological process during embryonic development, but it is thought to play an important role in the pathophysiology and treatment of many diseases, such as cancers (Kerr, Winterford, and Harmon 1994). As for blood vessels, apoptosis has been found only, so far, in neonatal lamb arteries (Cho, Courtman, and Langille 1995) and human incisal arteries (Thorball, Moe, and Winther-Nielsen 1985). Evidence has been accumulating, since 1994, to suggest that apoptosis probably occurs in hypertension (Devlin et al. 2000), peripheral and coronary atherosclerosis (Geng and Libby 1995), and cystal medial degeneration and aortic dissection (Ihling et al. 1999). Both endothelial cells and peripheral vascular smooth muscle cells (VSMCs) can undergo apoptosis in response to fibroblast growth factor and cyclic nucleotide analogues (Araki et al. 1990; Bennett, Evan, and Newby 1994).

It has been reported very recently that cocaine can induce apoptosis in cultured human coronary endothelial cells (He, Xiao, and Zhang 2001), hepatocytes (Wang et al. 2001), thymocytes (Wu et al. 2003), neuronal cells (Sharan et al. 2003), and rat testes (Li et al. 2003a). In this study, we hypothesized and found that cocaine can induce apoptosis in primary cultured aortic VSMCs. We found that cocaine can induce apoptosis in aortic VSMCs in a concentration-dependent manner; the greater the concentration of cocaine, the more rapid and the more intense was the apoptotic event. This may, thus, be one of the primary mechanisms of aortic dissection, atherosclerosis, and hypertension related to cocaine abuse.

MATERIALS AND METHODS

Cell Isolation and Primary Cell Culture

Smooth muscle cells from rat aortae were isolated according to an established method and cultured in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 1% l-gultamine (Zhang et al. 1992). Cells were cultured at 37°C in a humidified incubator composed of 95% air/5% CO₂ (Zhang et al. 1992). All the cells used for the experiments were passages 3 to 6 and were at 80% to 90% confluence. To confirm that cell death was occurring via apoptosis, cells were grown on coverslips. After 24 h of cell culture in serum-free medium, the primary cells were treated with cocaine HCl (10⁻⁶ to10⁻⁴ M) in the medium with 1% FCS for 12, 24, and 48 h, respectively.

Quantitative Morphological Analysis of Apoptosis

The cells were fixed with formaldehyde for 10 min, rinsed with phosphate-buffered saline (PBS) for 5 min, 0.2 ml of RNase A stock solution (1 mg/ml) was added at 37°C for 30 min, cells washed with PBS for 5 min, and then incubated with 10 μg/ml of propidium iodine or 10 μg/ml acridine orange for 15 to 20 min. After decolorization with distilled water, the cells were viewed with an ultraviolet fluorescence microscope and an ultraviolet laser confocal microscope. Cells with typical features of nuclear fragmentation and/or marked condensation of chromatin with reduced nuclear size were interpreted as apoptotic cells (He, Xiao, and Zhang 2001). The number of apoptotic cells and total cells were counted in six randomly selected high-power fields (at least 400 cells). The percentage of apoptotic cells was calculated as the number of apoptotic cells/number of total cells × 100%.

Statistics

Where appropriate, results are expressed as means ± SEM and were examined for statistical significance by means of Student t tests and analysis of variance (ANOVA). Values of p < 0.05 were considered to be statistically significant.

RESULTS

Propidium iodine or acridine orange was used to study the morphology of the nuclei. Cocaine caused nuclear changes characteristic of apoptosis in primary cultured rat aortic VSMCs (Figure 1). These included smaller nuclei and condensed chromatin compared to normal nuclei. Apoptotic bodies, the predominant morphological characteristic of apoptosis, were clearly evident. The percentage of apoptotic cells was increased by cocaine (10⁻⁶ to 10⁻⁴ M) at 24 h in a concentration-dependent manner (Figure 2), with significant increases even at the lowest concentration tested. At 10⁻⁴ M cocaine, apoptotic VSMCs were twofold more prevalent than in unexposed cells. Aortic VSMCs treated with 10⁻⁵ M cocaine for 12, 24, and 48 h all showed increased apoptotic rates compared with controls (Figure 3). In addition to being concentration-dependent, apoptosis was also time-dependent, with an increasing percentage of VSMCs undergoing apoptosis between 12 and 48 h.

DISCUSSION

The results presented here demonstrate apoptosis in VSMCs exposed to cocaine. Although apoptosis has been thought to play an important role in blood vessel dynamics, only during the past 10 years have researchers begun to show some interest in the possible role of apoptosis in vascular diseases. Apoptotic VSMCs were found in specimens of patients with cystic medial necrosis (CMN) (Ihling et al. 1999), aortic dilation associated with valvular disease (Bonderman et al. 1999), chronic aneurysm of the thoracic aorta, and dissection of the thoracic aorta (Lesauskaite et al. 2001). Nagashima et al. reported that the angiotensin II type 2 receptor can mediate VSMC apoptosis in cystic medial degeneration associated with Marfan’s syndrome (Nagashima et al. 2002). So apoptosis may play an important role in the pathogenesis of aortic dissection. During the past decade, physicians have noted that some patients diagnosed with aortic dissection often appeared to have a history of using cocaine during the 24 h before admission to the emergency room, and this phenomenon is more apparent in urban medical centers (Hsue et al. 2002). Cocaine seems to provoke dissection rarely, but, when it does, it strikes in the descending aorta and afflicts predominantly young, black, hypertensive individuals.

Possible mechanisms involved in cocaine-induced aortic dissection may include (1) cocaine inhibition of the re-uptake of both epinephrine and norepinephrine at the neural synapses, which leads to profound sympathetic stimulation, presumably causing such sheer stress on the aorta’s intima to produce a small “nick” or tear (Nallamothu et al. 2001); (2) cocaine could induce atherosclerosis; (3) depletion of [Mg²⁺]_i as happens in organs such as the brain (Altura and Gupta 1992) and VSMCs (Altura et al. 1993); and (4) enhanced cellular uptake of Ca²⁺ as happens in coronary and cerebral blood vessels (He et al. 1993; Lai et al. 2002). Our study clearly reveals that cocaine can also cause apoptosis of aortic VSMCs, which may be one of the primary mechanisms for cocaine-induced aortic dissection.

Apoptotic VSMCs can be found in coronary or peripheral atherosclerosis (Geng and Libby 1995), restenosis (Bennett, 1997), and hypertension (Devlin et al. 2000). This may help to explain why the number of VSMCs remains at the same level at 2 weeks and 3 days after angioplasty, despite the very active proliferation of VSMCs after endothelial injury. Apoptosis may also be one of the factors that contribute to the rupture of plaques (Bennett 1997). Some recent in vitro experiments suggest that aortic or coronary VSMCs can undergo apoptosis if induced by NO, peroxynitrite, H₂O₂, platelet-derived growth factor (PDGF), low serum, c-myc gene trans-fection, or angiotensin II, which suggests that apoptosis of at least peripheral VSMCs is regulated by many factors in vivo, thus potentially playing roles in the pathogenesis of several vascular diseases (Bennett, Evan, and Newby 1994; Pollman et al. 1996; Li et al. 2003b, 2004). Our study demonstrates, for the first time, that primary cultured aortic VSMCs can also undergo rapid apoptosis when treated with cocaine, and that this occurs in a concentration-dependent manner. We already know that chronic cocaine use may lead to atherosclerosis, strokes, and hypertension. Cocaine-induced apoptosis may, thus, also be one of the primary mechanisms causing atherosclerosis, hypertension, and strokes.

Cocaine-induced apoptosis has been shown in different cell types, including endothelium (He, Xiao, and Zhang 2001), thymocytes (Wu et al. 2003), neuronal cells (Sharan et al. 2003), and liver cells (Wang, 2001). The concentrations for inducing apoptosis of these different cells appear to be very similar to those reported herein. Although the mechanism of cocaine-induced apoptosis still remains unclear, cocaine-induced VSMC apoptosis may play an important role in cocaine-induced aortic dissection, atherosclerosis, and hypertension. If this is true, our findings may be important in designing new therapeutic drugs to ameliorate these diverse vascular-related problems.