Effect of Environmental Tobacco Smoke on LDL Accumulation in the Artery Wall
Background Previous research has shown that exposure to environmental tobacco smoke (ETS) increases the risk of atherosclerosis. To test the hypothesis that exposure to ETS increases LDL accumulation in the artery wall, we developed a model to measure the rate of LDL accumulation in individually perfused rat carotid arteries after the artery had been perfused with plasma taken from rats exposed to ETS (ETS-plasma).
Methods and Results Rats were exposed to ETS in a chamber in which steady-state sidestream smoke was continuously circulating. After exposure, blood from the animals was collected. Carotid arteries from unexposed rats were perfused first with normal plasma containing fluorescently labeled LDL. Then, the same arteries (10 arteries from five rats) were perfused with ETS-plasma plus fluorescently labeled LDL. Photometric measurements were made during perfusion of the arteries with fluorescently labeled LDL, and rate of LDL accumulation (mV/min) and lumen volume (mV) (volume of fluorescently labeled LDL solution) were determined. Perfusion with ETS-plasma increased the rate of LDL accumulation (mean±SEM, 6.9±1.8 mV/min) compared with control (1.6±0.40 mV/min, P≤.02). LDL accumulation was primarily dependent on LDL interaction with ETS-plasma rather than the interaction of ETS-plasma with the artery wall. Also, ETS-plasma significantly increased lumen volume (43.3±5.1 mV) compared with control (35.1±4.4 mV, P≤.005).
Conclusions Exposure to ETS acutely increased LDL accumulation in perfused arteries. Repeated exposure to ETS may represent important early events in atherogenesis.
Environmental tobacco smoke is composed of sidestream smoke (85%) that is elicited from the burning end of a cigarette and exhaled mainstream smoke directly emitted into the air by the smoker.1 Many reports have shown that smoking tobacco cigarettes is a health hazard; however, the link between exposure to ETS and health problems was first officially recognized by the US Surgeon General and National Academy of Sciences in 1986. Since that time, a number of epidemiological studies have linked ETS with heart disease.2 3 For example, Steenland4 reported that the risk of death due to cardiovascular diseases increases by 30% in nonsmokers who live with smokers. Furthermore, a recent report on heart disease and ETS suggests that more than 50 000 ischemic heart disease deaths in the United States are associated with ETS.5 However, little is known about the pathophysiological mechanisms of the development of atherosclerotic vascular disease induced by inhalation of ETS.
Zhu et al6 and Penn et al7 found in animal studies that inhalation of sidestream smoke from cigarettes promoted atherosclerotic plaque development. Additionally, ETS has been shown to lower HDLs in preadolescent children.8 These observations suggest that exposure to ETS may influence interactions of lipoproteins with the artery wall and promote atherosclerosis. Although atherosclerotic plaque development could be initiated or promoted by a number of factors, a cardinal feature of atherosclerotic lesions is the accumulation of LDL cholesterol within the artery wall. LDL is the major cholesterol-carrying lipoprotein present in humans. Accumulation of LDL in the artery wall has been strongly correlated with the development of atherosclerosis in both basic and clinical studies.
We hypothesized that exposure to ETS increased LDL accumulation in the artery wall. To test this hypothesis, we used an artery perfusion model in which LDL accumulation was measured in real time. Rats were exposed to ETS, and the plasma from these exposed animals was perfused into arteries of unexposed animals. The rate of LDL accumulation and lumen volume were measured by quantitative fluorescence microscopy. Our results demonstrate that short exposures to ETS increased artery wall accumulation of LDL and dilated these conductance arteries.
Sprague-Dawley rats (350 to 400 g) were received from Charles River Laboratories and maintained in a facility approved by the Animal Use Committee at UC Davis. Animals received rat chow and water ad libitum. On the day of the experiment, animals were transported in their own cages to the smoking chambers located at the Institute of Toxicology and Environmental Health on the UC Davis campus.
Fluorescently Labeled Molecules
Blood was obtained from human volunteers by venipuncture in tubes containing 15 mg EDTA. LDL was isolated by ultracentrifugation and labeled with the fluorescent hydrocarbon probe DiI as described by Pitas et al.9 The spectral properties of DiI are excitation maximum, 540 nm and emission maximum, 556 nm. Like rhodamine fluorescent probes, DiI resists quenching by the excitatory light. Previous studies showed that DiI binds avidly to lipoproteins and does not affect LDL binding capacity.10
To examine artery wall flux of reference nonlipid molecules, we perfused fluorescently labeled dextran (Sigma). Dextran (76 000 MW) was labeled with TRITC. The spectral properties of TRITC are excitation maximum, 554 nm and emission maximum, 573 nm.
The ETS exposure chambers at UC Davis have been described in detail previously.11 The parameters for the concentration of smoke were relative humidity, 45±0.5%; temperature, 23±1°C; carbon monoxide, 18±2 ppm; nicotine, 615±13 μg/m3; and total suspended particulates, 3.3±0.1 μg/m3.
On each day of the experiment, five rats received short-term exposure to ETS. Exposures were of 2 or 4 hours' duration. After smoke exposure, the rats were anesthetized with sodium pentobarbital (35 mg/kg IP). After the animals were deeply anesthetized, the chest cavity was exposed, and as much blood as possible was drawn by cardiac puncture through the left atrium. This procedure was lethal in all animals. All blood was collected in syringes containing streptokinase (50 000 U) and immediately placed on ice. Streptokinase has been shown not to alter the lipoprotein composition of the blood.12 The blood samples were centrifuged (2800 rpm for 20 minutes). Plasma was separated from red blood cells and filtered (0.2 μm Acrodisc serum syringe filters). After plasma was obtained from rats exposed to ETS, blood was drawn from five normal rats, and plasma was separated in an identical fashion. Thus, normal plasma and ETS-plasma from rats exposed to ETS for 2- or 4-hour exposures were available for artery perfusions.
Perfused Artery Protocol
Normal rats were anesthetized with sodium pentobarbital (35 mg/kg IP), and both carotid arteries were exposed and cannulated as described below. An incision was made in the proximal segment of the artery, and a cannula (23-gauge tubing adapter connected to polyethylene 50 tubing) was inserted into the carotid artery. The cannula was tied in place with 6-0 silk. Another incision was made in the distal portion of the artery before the bifurcation of the internal and external carotid arteries. Another cannula (identical to the proximal cannula) was inserted into the distal carotid artery and tied in place. The artery was removed from the animal and placed in a perfusion chamber (see Fig 1⇓). Throughout the surgery, the artery was superfused with Krebs-Henseleit solution gassed with 95% O2/5% CO2 and perfused with Krebs-Henseleit solution+0.1% BSA gassed with 95% O2/5% CO2.
The perfused artery was situated in a superfusate chamber on the microscope stage. In this chamber, the artery was continuously bathed in Krebs-Henseleit solution. The Krebs-Henseleit solution traveled from a reservoir through a heating coil to the superfusate chamber. A thermometer placed in the superfusate chamber continuously measured the temperature of the superfusate solution. The temperature of the superfusate solution was adjusted by changing the temperature of the heating coil. The superfusate temperature was maintained at 37°C for all experiments.
As described below, we perfused one artery on each experimental rig simultaneously. The plasma and plasma+DiI-LDL solutions and their reservoirs, the pump, and the Windkessel chambers were shared between the two perfusion experiments. Thus, each artery was perfused with solutions from either plasma or the plasma+DiI-LDL reservoirs at the same flow and perfusion pressures.
The two experimental rigs present in the laboratory were used to perfuse rat carotid arteries simultaneously and measure arterial flux of LDL. For each animal, the right and the left carotid arteries were placed into fluid-filled clear Plexiglas chambers positioned for viewing on the microscope platforms. One rig used to measure LDL flux consisted of a Nikon MII upright microscope with a dual optical path tube. A Plan X4 objective (numerical aperture, 0.1) was mounted on the microscope head. Mounted vertically on the dual optical path tube was a Nikon P1 photometer. Mounted horizontally on the dual optical path tube was a Hamamatsu CCD camera. The fluorescence image was transmitted by the dual optical path tube to the photometer and low-light television camera. The photometer measured changes in fluorescence intensity and was connected to a chart recorder and IBM-compatible computer. Output from the camera was entered into the videocassette recorder and a high-resolution monitor. The other rig used to measure LDL flux consisted of an inverted Nikon microscope (Diaphot-TMD) (6.3× objective; numerical aperture, 0.2), beamsplitter, Dage low-light television camera, and Nikon P101 photometer and controller. With this rig, the artery was imaged from below the Plexiglas chamber.
Quantitative Fluorescence Microscopy
Previous investigations studied mechanisms of lipoprotein flux in frog and hamster mesenteric venular capillaries by quantitative fluorescence microscopy.13 14 15 We extended the techniques developed in those studies to rat carotid arteries. The method that we used for determination of LDL flux in the artery wall entails labeling LDL with the fluorescent probe DiI. Under the appropriate optical conditions, DiI is excited to emit photons. These photons are collected and quantified by a photomultiplier (photometer). The photometer converts the number of photons detected into millivolts. Our calibration experiments showed that photons emitted by DiI-LDL as measured in millivolts were linearly related to LDL cholesterol concentration (R2=.99, P<.05).
After the carotid artery was positioned on the microscope stage, the location at which fluorescence was to be measured was illuminated with transmitted light and brought into focus. Then the transmitted light was turned off, and a shutter was opened to a mercury light. This light passed through a filter cube specific for the fluorescent molecule that was to be excited. Photons emitted by the fluorophore were captured and quantified by the photometer mounted on the microscope.
Measurement of Rate of LDL Accumulation and Lumen Volume
Initially, the rat carotid artery was perfused with normal rat plasma and a baseline level of fluorescence intensity was determined (Fig 2A⇓). Then DiI-LDL was added to normal rat plasma in another reservoir and perfused into the artery in which fluorescence measurements were made. During perfusion with plasma+DiI-LDL, DiI-LDL may bind to endothelium and/or cross into the artery wall. After washout of the lumen with plasma only, any DiI-LDL that remained on the luminal surface or in the artery wall was termed If accumulation (Fig 2B⇓). Thus, If accumulation determined photometrically (in millivolts) is a measure of the number of fluorescent molecules that remain on the lumen surface and in the artery wall.
The lumen volume was estimated by measurement of fluorescence intensity (millivolts) in the measuring window immediately after the artery was filled with the plasma+DiI-LDL (If0; Fig 2A⇑). Fluorescence measurements made later during plasma+DiI-LDL perfusions reflect fluorescence within the lumen and in the artery wall. The perfusion flow rate remained constant during the entire course of the experiment. If the artery is perfused at constant flow and perfusate concentration remains the same, changes in If0 measure changes in the number of fluorescently labeled molecules in the lumen. Then, changes in If0 reflect changes in lumen volume and presumably in artery tone.
In our experiments, we alternately perfused normal plasma (nonfluorescent solution) with either normal plasma+DiI-LDL or ETS-plasma+DiI-LDL. The rate of LDL accumulation (If accumulation/min) and lumen volume (If0) were determined with every perfusion of normal plasma+DiI-LDL. The same concentration of DiI-LDL was added to the ETS-plasma as to the normal plasma, and repeat measurements of If0 and If accumulation were determined.
In other experiments, rats were not exposed to ETS but rather to constituents of ETS (for example, nicotine), or inhibitors or mediators of vascular wall function (eg, L-NMMA) were added to the ETS-plasma. In all cases, control measurements of rate of LDL accumulation and lumen volume were made before and after exposure of the artery to ETS-plasma or plasma containing compounds important in vascular wall function.
Chemicals and Materials
The composition of Krebs-Henseleit solution (in mmol/L) was NaCl 116, KCl 5, CaCl2·H2O 2.4, MgCl2 1.2, NH2PO4 1.2, and glucose 11. Nicotine (lot 110H3379); L-NMMA, an inhibitor of nitric oxide synthase (lot 84H4037); and uric acid (lot 5340461) were obtained from Sigma Chemical Co.
Each experiment consisted of perfusion of a single segment of either the right or left carotid artery of the rat. In the basic experiment, two to four perfusions of normal plasma+DiI-LDL were performed, and the rate of LDL accumulation and lumen volume (If0) were measured. These are the control LDL perfusions. Then, the artery received one or more treatments (eg, plasma from ETS-exposed animals or nicotine). During these treatments, repeat measurements of the rate of LDL accumulation and lumen volume were obtained during DiI-LDL perfusions. Therefore, each treated artery was compared with its own control. To give each artery and every treatment equal weight, the means of all measurements of rates of LDL accumulation and lumen volume during control LDL perfusions and with each treatment were determined. When only one treatment was performed, control and treatment measurements were compared by paired t test. In experiments in which the artery received multiple treatments, the data were analyzed with a one-way repeated-measures ANOVA. Significant effects were analyzed with the Student-Newman-Keuls multiple-comparison test. Differences were considered to be significant when P<.05. All data sets were tested for normality by the Kolmogorov-Smirnov test. If a normal distribution was not observed, nonparametric tests were used. The Wilcoxon signed-rank test was used to compare two groups of data.
Effects of ETS Exposure on the Vascular Wall
Rats received exposure to ETS for 2 and 4 hours. These exposure times were used because they simulate the conditions of nonsmoking humans exposed to ETS. The artery was alternately perfused with normal plasma (nonfluorescent solution) and normal plasma+DiI-LDL (fluorescent solution). The rate of LDL accumulation and lumen volume were determined in individual arteries perfused with normal plasma+DiI-LDL (0.057±0.0001 mg/mL cholesterol, 0.008±0.005 mg/mL triglyceride, and 0.044±0.004 mg/mL protein). Then the normal plasma+DiI-LDL was replaced with ETS-plasma+DiI-LDL (same concentration as perfused with normal plasma). The same artery was alternately perfused with normal plasma and ETS-plasma+DiI-LDL, and the rate of LDL accumulation and lumen volume were again determined. Thus, the rate of LDL accumulation and lumen volume were determined during perfusions of normal plasma+DiI-LDL and during perfusions of ETS-plasma+DiI-LDL in the same artery.
Eleven arteries were perfused with plasma from rats exposed to ETS for 2 hours. The rates of LDL accumulation at control (normal plasma+DiI-LDL) and after ETS-plasma+DiI-LDL were 1.05±0.65 and 2.25±0.7 mV/min, respectively. Because the rates of LDL accumulation after perfusion with ETS-plasma (2 hours) did not appear to be normally distributed, we analyzed the data with a Wilcoxon signed-rank test. Of the 11 arteries, the rate of LDL accumulation increased in 8 arteries and decreased in 3 arteries perfused with ETS-plasma. This nonparametric test showed a significant increase in the rate of LDL accumulation during perfusion with ETS-plasma compared with control (W=45; P<.04). Lumen volume (If0) increased from 39.9±3.8 mV at control to 48.3±3.3 mV during perfusion with ETS-plasma (P≤.04).
Perfusion of plasma from rats exposed to ETS for 4 hours resulted in an increased rate of LDL accumulation and an increase in lumen volume. Fig 2⇑ is an example of the fluorescence measurements made during perfusion of a single carotid artery with plasma+DiI-LDL (Fig 2A⇑) and ETS-plasma+DiI-LDL (Fig 2B⇑). In 10 perfused arteries, a significant difference in the rate of LDL accumulation was present in arteries during control LDL perfusions (1.6±0.4 mV/min) compared with perfusions with ETS-plasma (6.9±1.8 mV/min, P<.01). Lumen volume also increased after perfusion of ETS-plasma compared with control perfusions (43.3±5.1 versus 35.1±4.4 mV, P<.05). These changes in lumen volume were confirmed by measurement of the artery ID at If0 by our optical system. ID was 0.99±0.06 mm during control plasma+DiI-LDL perfusions and 1.04±0.04 mm after perfusion of ETS-plasma+DiI-LDL (P<.002).
We examined the time course of the rate of LDL accumulation and lumen volume after perfusion with ETS-plasma (4 hours). Measurements of rate of LDL accumulation and lumen volume were made with every perfusion of plasma+DiI-LDL. Then, plasma+DiI-LDL was replaced with ETS-plasma+DiI-LDL. For the next 80 minutes, plasma and ETS-plasma+DiI-LDL were alternately perfused. Again, measurements of rate of LDL accumulation and lumen volume were made with every perfusion of ETS-plasma+DiI-LDL. Thus, the data were not continuous. The mean changes in the rate of LDL accumulation and in lumen volume after ETS-plasma+DiI-LDL were measured at 20-minute time intervals. The maximal increase in the rate of LDL accumulation was observed at 40 to 60 minutes after initiation of perfusion with ETS plasma compared with control (13.0±2.5 and 1.6±0.4 mV/min, respectively). Also, a significant increase in the rate of LDL accumulation was observed at 60 to 80 minutes (6.0±2.25 mV/min). There was an increase in lumen volume at a time interval of 20 to 40 minutes compared with control (52±5.0 and 35.1±4.4 mV, respectively). In comparison, inflammatory modulators of endothelial layer permeability, such as histamine, exert their greatest effects early (<10 minutes) after treatment of the vessel.15 Changes in LDL flux over time after perfusion with ETS-plasma suggest different mechanisms of macromolecule accumulation in the vessel wall.
In the experiments described above, both the rate of LDL accumulation and lumen volume increased after perfusion with ETS-plasma+DiI-LDL. To determine whether the increase in the rate of LDL accumulation was due to an increase in lumen volume, a Pearson product-moment correlation was used to compare these two variables. The change in the rate of LDL accumulation was not related to the change in the lumen volume for each perfused artery when the artery was perfused with normal plasma+DiI-LDL and when the same artery was perfused with ETS-plasma+DiI-LDL (r=.47; P>.05).
In another series of experiments, DiI-LDL was added only to normal plasma (not to ETS-plasma) to examine the effect of ETS-plasma on the artery wall rather than on LDL. The artery was perfused alternately with normal plasma and normal plasma+DiI-LDL. Then, the normal plasma was replaced with ETS-plasma and the artery was alternately perfused with ETS-plasma (nonfluorescent solution) and normal plasma+DiI-LDL (fluorescent solution). Under these conditions, the rate of LDL accumulation in the artery wall was not increased compared with control (1.0±0.2 versus 0.7±0.2 mV/min, respectively; n=10 arteries; P≥.88). These data indicated that exposure of the artery to ETS-plasma did not significantly increase LDL accumulation in the artery wall. LDL had to directly interact with ETS-plasma before an increased rate of LDL accumulation was observed in the artery wall.
Effect of ETS-Plasma on Reference-Molecule Rate of Accumulation
Reference-molecule rate of accumulation was compared with the rate of LDL accumulation in arteries perfused with ETS-plasma. Reference nonlipid molecules are not expected to bind to elements of the artery wall. The rate of neutral dextran (76 000 MW; labeled with TRITC) accumulation was determined in six arteries at control and after perfusion of ETS-plasma. Dextran (3.8 mg/mL) was added to normal plasma, and the control rate of dextran accumulation was 1.4±0.3 mV/min. Then, dextran (same concentration) was added to ETS-plasma (4-hour exposure), and repeat measurements of rate of dextran accumulation were determined (2.5±0.5 mV/min; P≤.04). Thus, the rate of dextran accumulation increased 78% after perfusion of ETS-plasma compared with control perfusions. These experiments demonstrate an increase in the rate of dextran accumulation, which we assume to be due to changes in endothelial layer permeability. In comparison, the rate of LDL accumulation increased 400% after perfusion of ETS-plasma. We compared the effect of ETS-plasma on the rates of LDL and dextran accumulation by two-way ANOVA. Comparisons of control rates of LDL and dextran accumulation were not significantly different. However, perfusion of ETS-plasma significantly increased the rate of LDL accumulation compared with the rate of dextran accumulation (P<.05).
Although LDL is a much larger molecule (2 500 000 MW) than dextran (76 000 MW), the relative change in the rate of LDL accumulation when arteries were perfused with ETS-plasma greatly exceeded that of dextran. These experiments suggest that increased LDL accumulation in the artery wall resulted from increased binding of LDL to the artery wall rather than increased LDL permeability.
Effect of Nicotine on the Rate of LDL Accumulation and Lumen Volume
Because nicotine has been shown to increase macromolecule permeability in cell culture studies, we examined the effect of nicotine on the rate of LDL accumulation in perfused arteries. DiI-LDL initially was added to normal plasma, and rate of LDL accumulation in each artery was determined. Then, nicotine was added to the normal plasma+DiI-LDL. We tested nicotine concentration in a dose comparable to that found in rats or humans exposed to ETS. The nicotine concentration found in rat plasma exposed to ETS under the same exposure conditions was 8 to 10 ng/mL.16 The rate of LDL accumulation during control DiI-LDL perfusions was 0.9±0.3 mV/min. When nicotine (10−8 ng/mL) was perfused into arteries, the rate of LDL accumulation was 1.4±0.8 mV/min (n=12 arteries; P≥.25). Thus, nicotine induced a small increase in artery wall LDL accumulation that was not statistically significant. Also, nicotine at the same dose had no effect on lumen volume (control, 36.8±2.2 mV versus nicotine-treated, 35.5±2.4 mV; P≥.32).
Mechanisms of ETS-Mediated Increases in Lumen Volume
We investigated the effects of both endogenous and exogenous nitric oxides on artery vasomotor tone. Nitrogen oxides (eg, NO, NO2) are present in tobacco smoke and could directly stimulate smooth muscle relaxation. Previous studies have shown that uric acid decreases lipid peroxidation and the formation of 3-NO2-Tyr and dityrosine.17 18 We examined the effect of uric acid on ETS-plasma–induced arterial dilation by first measuring lumen volume during control perfusions with plasma+DiI-LDL. Uric acid (10−3 mol/L) was added to the plasma+DiI-LDL, and lumen volume was measured again. Then, uric acid (same concentration) was added to the ETS-plasma+DiI-LDL, and lumen volume was determined. Changes in lumen volume for plasma+DiI-LDL, uric acid+plasma+DiI-LDL, and uric acid+ETS-plasma+DiI-LDL were 42.9±0.9, 39.6±0.96, and 38.3±1.0 mV, respectively (n=10 arteries). Thus, uric acid prevented ETS-plasma–induced arterial dilation.
To determine the role of endothelium-derived nitric oxide production, L-NMMA, an inhibitor of nitric oxide synthase, was tested in our system. Lumen volume during control perfusions of DiI-LDL was 42.8±1.8 mV (n=10 arteries). Then, L-NMMA (10−6 mol/L) was added to the plasma+DiI-LDL, and lumen volume was 43.6±2.3 mV. L-NMMA (same concentration) was added to ETS-plasma, and lumen volume was determined (55.8±5.6 mV). Lumen volume during treatment with ETS-plasma+L-NMMA was significantly greater than control or L-NMMA–treated lumen volume (P<.05). Control and L-NMMA lumen volumes were not significantly different. These experiments showed that inhibition of nitric oxide synthase had no effect on vasodilation associated with ETS-plasma.
Our studies showed that after a single exposure to ETS, LDL accumulation in the artery wall increased significantly. The increased LDL accumulation appears to be mediated primarily by increased binding of LDL to the artery wall rather than a change in artery wall permeability. LDL must have direct interaction with ETS-plasma for increased LDL accumulation to occur. This effect suggests biochemical modification of LDL by exposure to ETS-plasma.
The study of the effect of ETS on systemic arteries is difficult because of the complexity of the composition of ETS and the multiplicity of interactions ETS has with the respiratory system, blood, and artery wall. ETS is a complex mixture containing thousands of components within gaseous, vaporous, and particulate phases. ETS is filtered and processed in both large and small airways, enters the alveolar space, and crosses the respiratory epithelium to enter the pulmonary vascular system. Components of ETS may interact with the blood (for example, platelets or lipoproteins). Finally, processed components of ETS interact with the vascular wall. A model to study effects of ETS on the artery wall must account for the constituents of ETS and the complexity of processing of ETS by the respiratory tract, blood, and vascular wall. We focused our investigation on the interactions of ETS-plasma with the artery wall. Although it would have been much easier to bubble ETS through plasma, this approach does not consider the extensive processing of ETS in the respiratory system. Thus, by taking blood from rats exposed to ETS, we were able to directly study the interaction of ETS-plasma with the artery wall.
LDL accumulation in the arterial wall could be caused by an increase in LDL influx into the artery wall14 15 17 and/or by an increase in LDL retention and decrease in LDL efflux from the artery.18 19 Our studies were designed to examine the effect of acute exposure of ETS-plasma on LDL flux in the artery wall. Changes in endothelial layer permeability appeared to play a relatively small role in increasing LDL accumulation in the artery wall. Our studies showed a comparatively much greater rate of LDL accumulation than reference molecule (dextran) accumulation after perfusion of ETS-plasma. Furthermore, LDL that was not directly exposed to ETS-plasma showed no increase in LDL accumulation after the artery was perfused with ETS-plasma.
Previous studies in cell culture showed that nicotine increased macromolecule transendothelial transport.20 Our studies demonstrated a 56% increase in LDL accumulation when nicotine was added to the perfusate. However, this effect was not statistically significant. It is possible that if the sample size had been larger or the nicotine concentration higher, significant differences might have been seen. In comparison, the nicotine effect was much smaller than the effect of ETS-plasma on the rate of LDL accumulation. In other studies, similar results were found in the respiratory system. Nicotine at concentrations similar to ours did not increase airway plasma exudation, whereas cigarette smoke induced plasma exudation.21
Both endogenous and exogenous nitrogen oxides are reported to be important modulators of blood vessel tone as a result of exposure to cigarette smoke.22 Our studies did not implicate stimulation of endothelium-derived nitric oxide production by ETS-plasma, because an inhibitor of nitric oxide synthase had no effect on the ETS-induced artery dilation. However, dilation of the arteries was completely abolished with uric acid. Other studies have shown uric acid to be a scavenger of NO2. Uric acid is rapidly depleted when plasma is exposed to this gas.23 The inhibitory effects observed with uric acid are consistent with free-radical mechanisms of both 3-NO2-tyrosine and dityrosine formation by cigarette smoke.24
Conclusions and Implications
Our studies show that even short exposures to ETS increase artery wall accumulation of LDL. Increased LDL accumulation in the artery wall could be an initiating event in atherogenesis. These effects could be important in the home or workplace and have public policy implications for regulation of ETS. The effects of repeated intermittent exposure to ETS need further study. Finally, these studies demonstrate a potential mechanism by which ETS could facilitate atherogenesis, the major cause of adult morbidity and mortality in the United States.
Selected Abbreviations and Acronyms
|ETS||=||environmental tobacco smoke|
|ETS-plasma||=||plasma from animals exposed to ETS|
|If0||=||If immediately after artery was filled with plasma+DiI-LDL|
|UC||=||University of California|
This work was supported by the Tobacco-Related Disease Research Program of the University of California (3RT-0180 and 4RT-0213), the National Heart, Lung, and Blood Institute (K11-HL-02112), and the American Heart Association, California Affiliate (92-236). We are indebted to Kristine Lewis, Mable Woo, and Mike Goldsmith for their careful technical assistance. Charles O'Neil, PhD, and Steve McCurdy, MD, provided valuable comments about the manuscript.
- Received December 13, 1995.
- Revision received May 9, 1996.
- Accepted May 20, 1996.
- Copyright © 1996 by American Heart Association
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