Abstract
In cigarette smokers endothelial dysfunction, measured by flow-mediated dilation (FMD), precedes cardiovascular disease (CVD) and can be improved by supplementation with n - 3 polyunsaturated fatty acids (PUFAs). We developed a mouse model of cigarette smoke (CS)-induced endothelial dysfunction that resembles impaired FMD observed in human cigarette smokers and investigated the mechanism by which n - 3 PUFAs mediate vasoprotection. We hypothesized that loss of nitric oxide (NO)-dependent vasodilation in CS-exposed mice would be prevented by dietary n - 3 PUFAs via a decrease in oxidative stress. C57BL/6 mice were fed a chow or n - 3 PUFA diet for 8 weeks and then exposed to mainstream CS or filtered air for 5 days, 2 h/day. Mesenteric arterioles were preconstricted with U46619 and dilated by stepwise increases in pressure (0–40 mmHg), resulting in increases in flow, ± inhibitor of NO production or antioxidant, Tempol. Markers of oxidative stress were measured in lung and heart. CS-exposed mice on a chow diet had impaired FMD, resulting from loss of NO-dependent dilation, compared with air exposed mice. Tempol restored FMD by normalizing NO-dependent dilation and increasing NO-independent dilation. CS-exposed mice on the n - 3 PUFA diet had normal FMD, resulting from a significant increase in NO-independent dilation, compared with CS-exposed mice on a chow diet. Furthermore, n - 3 PUFAs decreased two CS-induced markers of oxidative stress, 8-epiprostaglandin-F2α levels and heme oxygenase-1 mRNA, and significantly attenuated CS-induced cytochrome P4501A1 mRNA expression. These data demonstrate that dietary n - 3 PUFAs can protect against CS-induced vascular dysfunction via multiple mechanisms, including increasing NO-independent vasodilation and decreasing oxidative stress.
Keywords: cigarette smoke, flow-mediated dilation, omega-3 polyunsaturated fatty acids, oxidative stress, vasodilation.
Cigarette smoke (CS) is a major independent risk factor for development of cardiovascular disease (CVD) (O’Donnell and Elosua, 2008), including atherosclerosis, myocardial infarction, aortic aneurysms, and stroke (Kawachi et al., 1997; Naya et al., 2008; Shinton and Beevers, 1989; Wilmink et al., 1999). Endothelial dysfunction, which is the loss of vasodilators within the vasculature, is the earliest pathological event in the development of atherosclerosis. We and others have shown that flow-mediated dilation (FMD), a clinical assessment of endothelial dysfunction, is impaired in smokers (Celermajer et al., 1993, 1996; Ozaki et al., 2010; Taylor et al., 2016; Wiest et al., 2015), that impaired FMD is a predictor of future cardiovascular events (Shechter et al., 2009; Witte et al., 2005), and that smoking cessation can significantly improve impaired FMD (Celermajer et al., 1993; Wang and Widlansky, 2009; Johnson et al., 2010).
Nonetheless, although smoking cessation programs have been established, successful cessation is difficult to achieve. According to the national health interview surveys between 2001 and 2010, nearly 70% of adult smokers in the U.S. showed an interest in cessation, about 50% made an attempt to quit, and yet only 6% successfully stopped smoking for more than 6 months (Centers for Disease Control and Prevention, 2011; Yong et al., 2014). Thus, the cardiovascular benefits derived from successful cessation could take years to achieve, highlighting the need for therapeutic strategies that help to reduce the development and progression of CS-induced CVD.
CS is a source of reactive oxygen species (ROS), such as superoxide anion and peroxynitrite. ROS present in CS can increase the levels of lipid peroxidation, as measured by thiobarbituric acid reactive substances (TBARS) (Miller et al., 1997; Morrow et al., 1995) as well as increases in other biomarkers of oxidative stress, such as 8-epiprostaglandin-F2α (8-isoprostane) and 8-hydroxydeoxyguanosine (8-OHdG) (Seet et al., 2011). Additionally, ROS present in CS can cause endothelial dysfunction by decreasing nitric oxide (NO) bioavailability (Barua et al., 2003). Studies have shown that impaired FMD assessed in conduit vessels is, at least in part, due to loss of NO-mediated dilation (Green et al., 2014; Kooijman et al., 2008).
One potential therapy for preventing CS-induced impaired FMD is supplements containing omega-3 polyunsaturated fatty acids (n - 3 PUFAs). The n - 3 PUFAs, eicosapentaenoic acid (EPA, 20:5n - 3) and docosahexaenoic acid (DHA, 20:6n - 3), are derived from the shorter chain precursor, α-linolenic acid (ALA, 18:3n - 3), and have been shown to be cardioprotective (Marik and Varon, 2009; Oh et al., 2014; Shimokawa and Vanhoutte, 1988). Although the mechanism of action of n - 3 PUFAs are not fully understood, they have been shown to be anti-inflammatory, antithrombotic, and vasodilatory agents (Abeywardena and Patten, 2011; Kris-Etherton et al., 2002). Furthermore, studies specifically in smokers show that consuming n - 3 PUFA supplements significantly improves FMD (Siasos et al., 2013).
The aim of this study was to develop a mouse model of CS-induced endothelial dysfunction that resembled impaired FMD observed in human cigarette smokers. This would enable us to mechanistically study the beneficial vascular properties of n - 3 PUFAs and further elucidate the therapeutic potential of n - 3-PUFAs to delay or prevent CS-induced CVD in smokers. We hypothesized that n - 3 PUFAs protect against CS-induced vascular dysfunction by increasing NO-bioavailability and decreasing oxidative stress. To test this hypothesis, we fed C57BL/6 mice an n - 3 PUFA-enriched diet or standard chow diet and then exposed the mice to mainstream CS or filtered air. We assessed markers of oxidative stress, the contribution of NO and ROS to FMD and the ability of n - 3 PUFAs to improve oxidative stress and FMD in CS-exposed mice.
MATERIALS AND METHODS
Chemicals
Nω-nitro-l-arginine (LNNA) and 4-hydroxy-TEMPO (Tempol) and all components of physiological saline solution (PSS) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-PSS were purchased from Sigma–Aldrich (St Louis, MO). U46619 was purchased from Cayman Chemical Company (Ann Arbor, MI) and ionomycin was purchased from EMD Millipore (Billerico, MA).
Animals and Diet
Seven-week-old male C57Bl/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were housed in a temperature-controlled environment, fed standard chow (2020X Rodent Diet, Teklad Diets, Envigo, Indianapolis, IN) and provided water ad libitum. After one week of acclimation, mice were randomly divided into two groups. One group continued to be fed the chow diet, whereas the second group was placed on an n - 3 PUFA-enriched diet (TD.110516; 150 g oil/kg diet) (Teklad Diets) for 8 weeks. The 8-week feeding was chosen because it has been established that RBC concentration is a reasonable surrogate of tissue concentrations and in humans EPA and DHA reach steady state in RBCs after 8–12 weeks of supplementation (Browning et al., 2012). Additionally, in most randomized control trials it has been shown that n - 3 PUFA supplementation significantly improves vascular function after 4–8 weeks (Wang et al., 2012). Thus, we chose 8 weeks of n - 3 PUFA dietary enrichment as a reasonable duration for ensuring > 80% steady state incorporation into tissues and for improving vascular function. Nutrients and fatty acid composition, and calories contained in the standard chow and n - 3 PUFA-enriched diets have been reported previously (Wiest et al., 2016).
CS Exposure
Mice from each diet were randomly assigned to either CS-exposed or air-exposed groups (n = 4–7/group). Mice were exposed to whole-body mainstream CS generated from University of Kentucky 3R4F reference cigarettes (9.4 mg tar, 0.73 mg nicotine per cigarette) in the SCIREQ InExpose system (SCIREQ, Montreal, Canada). Up to 5 mice were concurrently exposed to CS for 2 h/day (9 cigarettes total) for 5 consecutive days. Standard parameters, as set by the International Standard Organization (ISO 1991), were a 35 mL puff that lasted for 2 s followed by 58 s of fresh airflow at 6 mL/s. Average carbon monoxide (CO) levels were 181 ppm and average total particulate matter (TPM) was 271 mg/m3 (Figure 1). Immediately prior to sacrifice, the spontaneous void of urine was collected on a sheet of parafilm when the mice were removed from their cage. Mice were sacrificed 4 h following the last CS-exposure. The 4 h following the last CS-exposure was chosen to investigate the effect of CS exposure on vascular function without the acute effects of nicotine or cotinine. Other investigators have shown that plasma nicotine and cotinine levels in C57Bl/6 mice are significantly reduced 4 h post exposure (Siu and Tyndale, 2007; Zhou et al., 2010). Siu and Tyndale (2007) further showed that the t1/2 for nicotine is 9.2 min, whereas the t1/2 for cotinine is 23.7 min. Therefore, after 4 h, there would be < 0.0001% nicotine and 0.09% cotinine remaining in the circulation. Mice were administered an intraperitoneal (ip) injection of sodium heparin, anesthetized with ip injection of ketamine/xylazine (80/4 mg/kg) and euthanized by exsanguination. Blood was collected by cardiac puncture, using syringes containing EDTA. Plasma and packed RBCs were collected from whole blood by centrifugation. The heart was harvested, atria were dissected and total left ventricle was weighed and recorded. Liver, lung and kidney weights were also recorded. Intestine was excised and immediately placed in ice cold HEPES-PSS (0.13 M NaCl, 0.006 M KCl, 0.001 M MgCl2, 0.002 M CaCl2, 0.01 M HEPES, 2.6 × 10−5 M EDTA, and 0.01 M glucose, pH 7.5). Tissue, urine, RBCs, and plasma were stored at -80 °C. All animal protocols were approved by the University of New Mexico Animal Care and Use Committee (IACUC protocol 15-200331-HSC) and the investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
FIG. 1.
Carbon monoxide (CO) and total particulate matter (TPM) levels over the duration of the study.
Urinary Cotinine
Urinary cotinine was measured using an ELISA kit (Abnova, Taipei City, Taiwan) and analyzed by using a cotinine standard curve.
Gene Expression Analysis
Total RNA was isolated from heart and lung tissue, using the RNeasy kit (Qiagen, GmbH, Germany). cDNA was synthesized using iScript Select cDNA Synthesis Kit (Bio-Rad, Hercules, CA). The reaction mixture used for the qPCR amplification consisted of iQ SYBR Green Supermix, 250 pg cDNA/μL and 500 nM cytochrome P4501A1 (Cyp1a1), heme oxygenase 1 (Hmox1), NAD(P)H quinone dehydrogenase 1 (Nqo1) or DNA polymerase 2 (Pol2) sense and antisense primers (Table 1). An iCycler (Bio-Rad) was used to perform the qPCR amplification. Cycle threshold data for both the target gene of interest and control normalization gene, Pol2, were used to calculate mean normalized expression.
TABLE 1.
Real Time PCR Primer Sequences
Gene | Sense Primer | Antisense Primer |
---|---|---|
Cyp1a1 | 5ʹ GGAACTAGACACAGTGATTG 3’ | 5’ TTGGGGATATAGAAGCCATTC 3’ |
Nqo1 | 5’ CCTTTCCAGAATAAGAAGACC 3’ | 5’ AATGCTGTAAACCAGTTGAG 3’ |
Hmox1 | 5’ CATGAAGAACTTTCAGAAGGA 3’ | 5’ TAGATATGGTACAAGGAAGCC 3’ |
Pol2 | 5’ TGACTCACAAACTGGCTGACATT 3’ | 5’ TACATCTTCTGCTATGACATGGG 3’ |
RBC n - 3 and n - 6 PUFAs
Packed RBCs obtained from EDTA-treated whole blood were analyzed for fatty acid content by OmegaQuant (Sioux Falls, SD) as previously described (Harris et al., 2013). Briefly, samples were heated with methanol and boron trifluoride to generate fatty acid methyl esters. These methyl esters were extracted with hexane and water and analyzed using a GC2010 (Shimadzu Corporation, Columbia, MD) gas chromatographer equipped with a 30 m capillary column (Omegawax 250, Supelco, Bellefonte, PA). Fatty acids were identified by comparison to standard fatty acid methyl esters.
Biomarkers of Oxidative Stress
All analyses were conducted in heart and lung tissue homogenates. All samples were flash-frozen in liquid nitrogen upon harvesting and all assays were conducted per manufacturer’s directions. A TBARS assay kit (Cayman Chemical) was utilized to investigate lipid peroxidation by measuring TBARS, a byproduct formed during decomposition of unstable peroxides derived from fatty acids, and expressed as malondialdehyde (MDA), based on a standard curve. Furthermore, the 8-isoprostane EIA kit (Cayman Chemical) was used to measure 8-epi PGF2α. Last, a glutathione assay kit (Cayman Chemical) was utilized to measure the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). Additionally, we analyzed levels of GSH and GSSG separately and there were no differences among groups.
Mesenteric Vasoreactivity Analysis
First order mesenteric arterioles were cleaned of fat and connective tissue and a leak-free segment was mounted in a pressure myograph chamber (DMT-110 systems, Danish Myo Technology, Ann Arbor, MI), containing PSS at 37 °C (0.13 M NaCl, 0.0047 M KCl, 0.0012 M KH2PO4, 0.0012 M MgSO4, 0.015 M NaHCO3, 2.6 × 10−5 M CaNa2-EDTA, 0.0055 M glucose, and 0.0018 M CaCl2, pH 7.4) and bubbled with 21% O2, 6% CO2 and balanced N2. Arterioles were equilibrated from 5 mmHg to 75 mmHg by increasing pressure in 10 mmHg increments every 5 min followed by 30 min at 75 mmHg. Baseline internal diameter was measured using edge detection software (MyoView Acquisition Software, DMTVAS 6.2.0.59 Danish Myo Technology). Arterioles were deemed viable if they constricted to a minimum of 30% of baseline internal diameter following KCl (0.06 M) treatment. For FMD experiments, arterioles were constricted to 45% of baseline diameter with U46619 (10−8 M) and then subjected to increases in flow by stepwise increases in pressure from 0 to 40 mmHg between inflow and outflow, while keeping mean pressure at 75 mmHg. The study was then repeated following 15 min incubation with LNNA (10−4 M). At the end of each study, passive internal diameter was determined using the calcium ionophore, ionomycin (10−5 M). There were no differences in passive internal diameter among groups (chow: air 185.6 ± 7.3; CS: 190.8 ± 6.8, n - 3 PUFA: air: 177.3 ± 6.1; CS: 184.4 ± 5.1; chow + Tempol: air: 172.1 ± 6.8; CS: 176.0 ±7.3). In addition, FMD experiments were conducted from air- and CS-exposed mice on a chow diet in the presence of Tempol (1 mM).
Statistical Analysis
All data were analyzed by SigmaPlot 13.0. Data are expressed as mean ± SEM, unless stated otherwise. FMD vascular reactivity pressure response data was analyzed by two-way repeated measures analysis of variance (ANOVA) with post hoc Holm–Sidak comparisons. Total area under the curve for vascular reactivity and all other data were analyzed by two-way ANOVA with post hoc Holm–Sidak comparisons. P < .05 was considered statistically significant.
RESULTS
CS Exposure Increased Urinary Cotinine Levels in Mice
Urinary cotinine (a metabolite of nicotine) was measured as an index of CS exposure. Mice exposed to CS exhibited increased urinary cotinine, compared with mice exposed to air on both a chow- and an n - 3 PUFA diet (chow diet: air: 130.4 ± 256.0 µg cotinine/g creatinine, CS: 4573.5 ± 269.9 µg cotinine/g creatinine, P < .001; n - 3 PUFA diet: air: 533.1 ± 256.0 µg cotinine/g creatinine, CS: 3459.0 ± 244.1 µg cotinine/g creatinine, P < .001).
CS Exposure Decreased Liver-to-Body Weight Ratio and the n - 3 PUFA Diet Attenuated This Decrease
Body and organ weights, and organ-to-body weight ratios were analyzed to determine if CS had any overt adverse effects and whether these effects were altered by an n - 3 PUFA diet. Body weights were not altered by CS on either diet (Table 2). However, CS significantly decreased liver-to-body weight ratio in mice fed a chow diet. While CS significantly decreased liver weight in mice fed the n - 3 PUFA diet, it failed to decrease liver-to-body weight ratio. An n - 3 PUFA diet decreased liver-to-body weight ratio in both CS- and air-exposed mice. Last, the n - 3 PUFA diet increased kidney weight in the air-exposed group and kidney-to-body weight ratio in the CS-exposed group, compared with mice on the chow diet.
TABLE 2.
Comparison of Body Weights of 4 Month Old C57BL/6 Male Mice on a Chow or n - 3 PUFA Diet and Exposed to Cigarette Smoke or Air for 5 Days
Chow Dieta |
n - 3 PUFA Diet |
|||
---|---|---|---|---|
Weight (g) | Air (n = 10) | Cigarette Smoke (n = 10) | Air (n = 15) | Cigarette Smoke (n = 15) |
Body | 26.9 ± 0.6 | 28.9 ± 0.6 | 30.6 ± 0.7 | 27.7 ± 0.7 |
Heart | 0.108 ± 0.003 (0.40 ± 0.01)b | 0.121 ± 0.003 (0.42 ± 0.01) | 0.116 ± 0.003 (0.38 ± 0.01) | 0.108 ± 0.003 (0.39 ± 0.01) |
Kidney | 0.280 ± 0.008 (1.04 ± 0.02) | 0.292 ± 0.008 (1.01 ± 0.02) | 0.325 ± 0.009# (1.07 ± 0.03) | 0.305 ± 0.009 (1.11 ± 0.03)# |
Lung | 0.145 ± 0.004 (0.54 ± 0.02) | 0.146 ± 0.004 (0.51 ± 0.02) | 0.150 ± 0.004 (0.50± 0.02) | 0.140 ± 0.004 (0.51 ± 0.02) |
Liver | 1.41 ± 0.06 (5.23 ± 0.10) | 1.28 ± 0.06 (4.63 ± 0.10)* | 1.408 ± 0.06 (4.58 ± 0.11)# | 1.19 ± 0.06* (4.27 ± 0.11)# |
Values are expressed as mean ± SEM. Data were analyzed by two-way ANOVA with Holm–Sidak post hoc analysis.
(Organ weight (g)/body weight (g)) × 100.
P < .05 versus air group fed the same diet.
P < .05 versus same treatment group fed the chow diet.
CS and an n - 3 PUFA Diet Significantly Altered RBC Fatty Acid Composition
To interpret physiological changes associated with CS exposure and the n - 3 PUFA diet, levels of fatty acids in RBCs of mice were analyzed. CS exposure decreased ALA levels and increased arachidonic acid (AA) levels in mice on a chow diet, compared with the air exposed group. Notably, both these CS-induced changes were prevented by the n - 3 PUFA diet (Table 3). Additionally, mice fed the n - 3 PUFA diet exhibited significantly higher percentages of EPA and DHA, and significantly lower percentages of linolenic acid (LA) and AA, compared with mice fed a chow diet. Because the n - 3 PUFA diet contained less of the n - 3 PUFA precursor, ALA, than the chow diet, mice fed the n - 3 PUFA diet exhibited significantly lower percentages of ALA.
TABLE 3.
RBC Fatty Acid Profile of 4 Month Old C57BL/6 Male Mice on a Chow or n - 3 PUFA Diet and Exposed to Cigarette Smoke or Air for 5 Days
Chow Dietb |
n - 3 PUFA Diet |
|||
---|---|---|---|---|
Fatty acid (%) | Air (n = 10) | Cigarette Smoke (n = 10) | Air (n = 15) | Cigarette Smoke (n = 15) |
n - 3 PUFAsa | ||||
ALA | 0.110 ± 0.004 | 0.092 ± 0.004* | 0.040 ± 0.003# | 0.039 ± 0.003# |
EPA | 0.228 ± 0.167 | 0.225 ± 0.167 | 10.1 ± 0.136# | 10.4 ± 0.136# |
DHA | 6.64 ± 0.16 | 7.04 ± 0.16 | 14.00 ± 0.13# | 13.80 ± 0.13# |
n - 6 PUFAs | ||||
LA | 12.5 ± 0.2 | 12.2 ± 0.1 | 2.3 ± 0.1# | 2.2 ± 0.1# |
AA | 16.5 ± 0.1 | 17.1 ± 0.1* | 8.4 ± 0.1# | 8.2± 0.1# |
Abbreviations: α-linolenic acid (ALA, 18:3n3), eicosapentaenoic acid (EPA, 20:5n3), docosahexaenoic acid (DHA, 22:6n3), linoleic acid (LA, 18:2n6), arachidonic acid (AA, 20:4n6).
Values are expressed as mean ± SE, n = 10–15. Data was analyzed by 2-way ANOVA with Holm–Sidak post hoc comparisons.
P < .05 versus chow air.
P < .05 versus same treatment group fed the chow diet.
CS Induced, and the n - 3 PUFA Diet Attenuated, Cyp1a1 Expression in the Heart and Lung
To further characterize the CS exposure and the influence of the n - 3 PUFA diet, the degree to which a 5-day CS exposure induced Cyp1a1 mRNA was investigated. As expected, CS significantly induced Cyp1a1 in both the heart and lung of mice on the chow and n - 3 PUFA diets (Figure 2A and B). However, the n - 3 PUFA diet significantly reduced both the basal and CS-induced levels of Cyp1a1 in the heart and reduced the basal levels of Cyp1a1 in the lung.
FIG. 2.
Effects of CS and an n - 3 PUFA diet on mRNA expression of Cyp1a1. A and B, Lung and heart mRNA expression of Cyp1a1, normalized to the housekeeping gene, Pol2, from air- and CS-exposed mice fed a chow or n - 3 PUFA diet. Data are shown as mean ± SE, n = 5–6/group and analyzed by two-way ANOVA with post hoc Holm–Sidak comparisons. *P < .05 versus air within the same diet; #P < .05 versus chow within the same exposure.
CS Induced One Biomarker of Oxidative Stress and the n - 3 PUFA Diet Prevented This Induction
To assess if oxidative stress biomarkers were induced by CS and influenced by the n - 3 PUFA diet, we measured the GSH/GSSG ratio, MDA and 8-isoprostane levels in the heart and lung. Neither CS nor the n - 3 PUFA diet affected GSH/GSSG levels in the heart or lung (Figure 3A and B). In addition, CS had no effect on MDA levels in the heart or lung, while the n - 3 PUFA diet significantly increased MDA levels in the heart (Figure 3C and D). Final, CS significantly induced 8-isoprostane levels in the heart and this induction was prevented in mice fed the n - 3 PUFA diet (Figure 3E). Although CS failed to induce 8-isoprostane levels in the lung on the chow diet, 8-isoprostane levels were significantly lower in the CS exposure group on the n - 3 PUFA diet (Figure 3F).
FIG. 3.
Effects of CS and n - 3 PUFA diet on oxidative stress biomarkers. Lung and heart levels of A and B, GSH/GSSG ratio, C and D, MDA levels, and E and F, 8-isoprostane levels from air- and CS-exposed mice fed a chow or n - 3 PUFA diet. Data are shown as mean ± SEM, n = 4–5/group and analyzed by two-way ANOVA with post hoc Holm–Sidak comparisons. *P < .05 versus air, within the same diet; #P < .05 versus chow within the same exposure.
CS and the n - 3 PUFA Diet Differentially Regulated Antioxidant Gene Expression in the Heart and Lung
To investigate the effect of CS and an n - 3 PUFA diet on antioxidant gene expression, mRNA expression of Hmox1 and Nqo1 were measured in the heart and lung. CS significantly induced Hmox1 in the heart and lung from mice fed the chow diet, and this induction was normalized to the air exposure levels in mice fed the n - 3 PUFA diet (Figure 4A and B). In contrast, CS significantly induced Nqo1 mRNA expression in the lung, but not the heart, and while this induction was attenuated by the n - 3 PUFA diet it was not restored to the air exposure levels (Figure 4C and D).
FIG. 4.
Effects of CS and an n - 3 PUFA diet on mRNA expression of Hmox1 and Nqo1. Lung and heart mRNA expression of A and B, Hmox1, normalized to the housekeeping gene, Pol2; and C and D, Nqo1, normalized to the housekeeping gene, Pol2, from air- and CS-exposed mice fed a chow or n - 3 PUFA diet. Data are shown as mean ± SE, n = 6–9/group and analyzed by two-way ANOVA with post hoc Holm–Sidak comparisons. *P < .05 versus air, within the same diet; #P < .05 versus chow within the same exposure.
CS Impaired FMD and the Antioxidant, Tempol, Prevented This Impairment by Increasing NO-Independent Dilation
To determine the degree to which this acute 5-day CS exposure impaired FMD and establish the contribution of oxidative stress and NO to this impairment, FMD studies were conducted in pressurized mesenteric arterioles from air- and CS-exposed mice fed a chow diet in the presence or absence of the free radical scavenger, Tempol, and the NO synthase inhibitor, LNNA. CS exposure significantly impaired total FMD, compared with air-exposed mice, and this impairment was completely prevented by Tempol (Figure 5A and C). By adding LNNA, the NO-dependent and -independent contribution to the FMD response could be determined from area-under-the-curve analysis. These analyses showed that the CS-induced impairment of FMD resulted from a significant reduction in NO-dependent dilation (Figure 5B and D). Interestingly, while Tempol tended to normalize the NO-dependent dilation response (P = 0.095), compared with no Tempol, it improved CS-impaired FMD by significantly increasing NO-independent dilation (Figure 5D).
FIG. 5.
Effects of CS and Tempol on FMD. A and B, Pressure response for FMD in pressurized mesenteric arterioles of air- and CS-exposed mice fed a chow diet with and without Tempol and in the presence and absence of LNNA, respectively. C, Area-under-the-curve analysis of the pressure-response curve shown in panel A. D, Area-under-the-curve analysis of the pressure-response curves shown in panel A and B, showing NO-dependent and NO-independent components of the FMD response. Data are shown as mean ± SEM, n = 4–7/group. Panel A and B: analyzed by two-way repeated measures ANOVA with post hoc Holm–Sidak comparisons. *P < .05 versus no Tempol, air; #P < .05 versus no Tempol, CS. †P < .05 versus no Tempol, air. Panel C and D, analyzed by two-way ANOVA with post hoc Holm–Sidak comparisons. *P < .05 versus air, no Tempol; #P < .05 versus no Tempol, CS.
n - 3 PUFA Diet Prevented CS Impairment of FMD by Increasing NO-Independent Dilation
To determine the degree to which an n - 3 PUFA diet could improve CS-impaired FMD and dissect the contribution of NO to this response, FMD studies were conducted in pressurized mesenteric arterioles from air- and CS-exposed mice fed an n - 3 PUFA diet in the presence or absence of LNNA. The impairment of the total FMD response induced by CS on a chow diet was completely prevented in mice fed an n - 3 PUFA diet (Figure 6A and B). Surprisingly, area-under-the-curve analysis in the presence and absence of LNNA revealed that the n - 3 PUFA diet prevented CS impaired FMD by significantly increasing NO-independent dilation without affecting NO-dependent dilation (Figure 6B and D).
FIG. 6.
Effects of CS and an n - 3 PUFA diet on FMD. A and B, Pressure response for FMD in pressurized mesenteric arterioles of air- and CS-exposed mice fed a chow or n - 3 PUFA diet in the presence and absence of LNNA, respectively. C, Area-under-the-curve analysis of the pressure-response curve shown in panel A. D, Area-under-the-curve analysis of the pressure response curves shown in panel A and B, showing NO-dependent and NO-independent components of the FMD response. Data are shown as mean ± SEM, n = 6–7/group. Panel A and B: analyzed by two-way repeated measures ANOVA with post hoc Holm–Sidak comparisons. *P < .05 versus chow, air; #P < .05 versus chow, air. Panel C and D, analyzed by two-way ANOVA with post hoc Holm–Sidak comparisons. *P < .05 versus chow, air; #P < .05 versus chow, CS.
DISCUSSION
In this study we established a FMD model that mimics CS-impaired FMD in humans (i.e. loss of total dilation and loss of NO-mediated dilation) and found that a diet enriched in n - 3 PUFAs can prevent CS-induced vascular dysfunction. However, the n - 3 PUFA diet appears to have multiple mechanisms of potential benefit. First, the n - 3 PUFA diet increases the contribution of NO-independent pathways to the dilation response with little-to-no effect on NO-dependent dilation. Second, the n - 3 PUFA diet acts an antioxidant as is evidenced by decreasing a specific marker of oxidative stress and the gene expression of a specific ROS-induced antioxidant enzyme. Final, the n - 3 PUFA diet reduces CS-induced expression of Cyp1a1, which has been associated with vascular dysfunction in earlier studies. Taken together these results demonstrate that an n - 3 PUFA-enriched diet can protect against CS-induced vascular dysfunction via multiple mechanisms, including NO-independent vasodilation and antioxidant effects.
The most novel finding of our study is that dietary n - 3 PUFAs protect against CS impaired FMD by improving NO-independent dilation. This result is unexpected since numerous earlier studies show that n - 3 PUFAs increase NO and NO bioavailability (Agbor et al., 2014; Li et al., 2007; Stebbins et al., 2008; Tagawa et al., 2002; Wu et al., 2012). For example, in a study measuring FMD in patients with coronary artery disease at baseline and after 3 months of EPA treatment (1800 mg/day), FMD is significantly improved following EPA treatment. In the presence of a NO-donor, sodium nitroprusside, there is no difference, showing that n - 3 PUFAs improve dilation, at least in part, by improving NO-mediated dilation (Tagawa et al., 2002). In another study in bovine aortic endothelial cells, EPA (25 µM) increases eNOS phosphorylation and NO production in an AMP-activated protein kinase (AMPK) dependent manner. Furthermore, acetylcholine-mediated dilation in eNOS-/- mouse aortic rings treated with EPA, is significantly impaired compared with ApoE-/- mice treated with EPA (Wu et al., 2012).
In our study, NO contributes approximately 50% to the FMD response in mesenteric arterioles in control mice. This is similar to the NO contribution to FMD and to acetylcholine-induced dilation in mouse mesenteric arterioles reported by others (Loufrani et al., 2001; Mendoza et al., 2010; Fujiwara et al., 2012) as well as to acetylcholine-induced dilation in rat mesenteric arterioles (Awe et al., 2006). Subsequently, we found that CS exposure reduced the NO contribution to FMD in mesenteric arterioles to 18%. This is similar to the reduction reported in acetylcholine-induced dilation in rat mesenteric arterioles following exposure to lipid-soluble smoke particles ex vivo (Zhang et al., 2006). Surprisingly, however, we found that the n - 3 PUFA diet significantly reduces the NO contribution to FMD in control mice and does not improve it in CS-exposed mice. Rather, the protective benefit of the n - 3 PUFA diet in CS-exposed mice is derived from a significant increase in the NO-independent contribution to FMD.
NO-independent dilation is primarily mediated by endothelial-derived hyperpolarizing factors (EDHFs). Although the exact mechanism of EDHF-mediated dilation is still unknown, P450-dependent metabolites of AA, DHA and EPA as well as potassium ions are proposed as possible EDHFs (Chen and Cheung, 1996; Edwards et al., 1998). In our study the ability of the n - 3 PUFAs to improve FMD may result from (1) shear stress-mediated activation of phospholipase A2 (PLA2), which releases membrane-bound fatty acids, including EPA and DHA (Pearce et al., 1996; Rosa and Rapoport, 2009), and (2) the subsequent metabolism of EPA and DHA by P450s to vasodilatory epoxides, epoxyeicosatetraenoic acids (EEQs) and epoxydocosapentaenoic acids (EDPs) (Lauterbach et al., 2002). EEQs and EDPs cause vasodilation through opening of large conductance, calcium-dependent potassium channels (BK channels) and ATP-sensitive potassium channels (Agbor et al., 2012; Hoshi et al., 2013; Walker, unpublished data), and thus act as EDHFs.
It is also notable and unexpected that the antioxidant Tempol similarly increases NO-independent dilation significantly. Tempol increases the NO-independent component of FMD from 33% to 55% in mice exposed to CS. Whereas FMD is largely thought to be NO-dependent (Rizzo et al., 1998; Tagawa et al., 2002), the mechanism of the FMD response may depend on vessel size where NO is the primary dilator in larger conduit vessels, while NO and EDHFs both contribute to FMD in smaller resistance vessels, like mesenteric arterioles. Nonetheless, our data suggest that oxidative stress may play a role in reducing NO-independent dilation. It is known that ROS can inhibit potassium channel function in the vasculature (Gutterman et al., 2005) and this is consistent with data showing that EDHF-mediated dilation is impaired in a rat model, in part, due to an increase in ROS (Leo et al., 2011).
A second major finding of our study is that the 5-day acute exposure to CS induces two specific markers of oxidative stress and these are attenuated by the n - 3 PUFA diet. It is well established that cigarette smokers have increased ROS, leading to an increase in markers of oxidative stress, such as GSH/GSSG ratio, MDA, and 8-isoprostanes (Jaimes et al., 2004; Seet et al., 2011). In our study, we observe an increase in two specific markers of oxidative stress, 8-isoprostane and mRNA expression of the antioxidant enzyme, Hmox1, following CS exposure. 8-Isoprostane is produced by the nonenzymatic oxidation of AA in membrane phospholipids and our data are consistent with studies showing elevated levels of plasma 8-isoprostane in heavy smokers (Morrow et al., 1995) and in nonsmokers exposed to secondhand smoke (Kato et al., 2006). Hmox1 is a gene induced by ROS via activation of the transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) and we also found that Hmox1 mRNA is increased in mice exposed to CS on a chow diet, consistent with other CS-exposed animal models (Wei et al., 2015; Yang et al., 2015). Importantly, we found that CS-induced increases in both 8-isoprostane levels and Hmox1 mRNA expression are reduced in mice fed an n - 3 PUFA diet, compared with those fed a chow diet. Whereas we did not observe any changes in the GSH/GSSG ratio or MDA levels in the heart and lung as a result of CS exposure, it is possible that GSH could be taken up from the plasma (Bai et al., 1994), while the lack of changes in MDA may result from nonspecific binding of thiobarbituric acid to other compounds in addition to lipids (Knight et al., 1988). Whereas we did observe that mice on the n - 3 PUFA diet exhibit increases in MDA in the heart, regardless of CS exposure, this is consistent with the findings of others (Gonzalez et al., 1992; Kawachi et al., 1997), and may result from the accumulation of DHA in the heart (Agbor et al., 2014; Arnold et al., 2010) and the increased susceptibility of the heart to oxidation (Santos et al., 2011).
The third major finding of our work is that the CS induction of Cyp1a1 is significantly attenuated by the n - 3 PUFA diet. Previous work shows that CYP1A1 is a risk factor for vascular dysfunction and hypertension in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Kopf et al., 2010b). Further, TCDD-induced increases in ROS is downstream of Cyp1a1 induction (Kopf and Walker, 2010a). Thus, CS-induced Cyp1a1 may contribute to the CS-induced increases in oxidative stress, while the attenuation of Cyp1a1 induction by the n - 3 PUFA diet may contribute to decreases in oxidative stress. The ability of the n - 3 PUFA diet to attenuate Cyp1a1 induction is consistent with previous studies of mice and rats treated with TCDD (Palaniswamy et al., 2014; Wiest et al., 2016); however, the mechanism underlying this attenuation remains to be investigated.
There are some potential limitations to this work. The 5-day CS exposure cannot be extrapolated to fully encompass the vascular injury and dysfunction that occurs following chronic CS exposure in humans. Nonetheless, our model of acute CS-exposure allows us to mechanistically investigate the early events that contribute to impaired FMD and the protection afforded by dietary n - 3 PUFAs in the impairment. Additionally, chronic cigarette smokers exhibit reduced levels of RBC EPA and DHA (Wiest et al., 2015), which likely contribute to impaired FMD. Thus, although the 5-day CS exposure is too short of an exposure time to reduce the levels of these n - 3 PUFAs, this limitation could be overcome by putting mice on a diet low in n - 3 PUFAs.
In summary, we show that loss of FMD in mesenteric arterioles of mice exposed to CS is prevented by an n - 3 PUFA diet, specifically by increasing NO-independent dilation and this protection is associated with decreases in Cyp1a1 induction and two specific markers of oxidative stress. These data provide preclinical mechanistic evidence that support clinical data that n - 3 PUFA supplements can improve impaired FMD in human cigarette smokers (Din et al., 2013; Siasos et al., 2013). Future studies are needed to elucidate the NO-independent mechanism by which n - 3 PUFAs afford their vasoprotection following CS exposure.
ACKNOWLEDGMENTS
The authors thank Dr Nancy Kanagy for her expertise in the vasoreactivity studies and Dr Matthew Campen for his assistance in CS exposures.
FUNDING
This work was supported by the National Institute of Environmental Health Sciences [R15ES021896 to M.K.W.] and by the National Heart Lung and Blood Institute [R15HL130970 to M.K.W.].
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