Tempol improves cutaneous thermal hyperemia through increasing nitric oxide bioavailability in young smokers

Abstract

We recently found that young cigarette smokers display cutaneous vascular dysfunction relative to nonsmokers, which is partially due to reduced nitric oxide (NO) synthase (NOS)-dependent vasodilation. In this study, we tested the hypothesis that reducing oxidative stress improves NO bioavailability, enhancing cutaneous vascular function in young smokers. Ten healthy young male smokers, who had smoked for 6.3 ± 0.7 yr with an average daily consumption of 9.1 ± 0.7 cigarettes, were tested. Cutaneous vascular conductance (CVC) during local heating to 42°C at a rate of 0.1°C/s was evaluated as laser-Doppler flux divided by mean arterial blood pressure and normalized to maximal CVC, induced by local heating to 44°C plus sodium nitroprusside administration. We evaluated plateau CVC during local heating, which is known to be highly dependent on NO, at four intradermal microdialysis sites with 1) Ringer solution (control); 2) 10 μM 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol), a superoxide dismutase mimetic; 3) 10 mM N^ω-nitro-l-arginine (l-NNA), a nonspecific NOS inhibitor; and 4) a combination of 10 μM tempol and 10 mM l-NNA. Tempol increased plateau CVC compared with the Ringer solution site (90.0 ± 2.3 vs. 77.6 ± 3.9%maximum, P = 0.028). Plateau CVC at the combination site (56.8 ± 4.5%maximum) was lower than the Ringer solution site (P < 0.001) and was not different from the l-NNA site (55.1 ± 4.6%maximum, P = 0.978), indicating the tempol effect was exclusively NO dependent. These data suggest that in young smokers, reducing oxidative stress improves cutaneous thermal hyperemia to local heating by enhancing NO production.

almost 6 million people die from tobacco use and exposure each year (53), and the majority of tobacco-related deaths are due to cardiovascular disease (11). Indeed, chronic exposure to cigarette smoking changes the structure and function of human conduit arteries (45). Oxidative stress is suspected to be a major contributor to chronic cigarette smoking-induced vascular alterations, as reducing oxidative stress with antioxidants (e.g., vitamin C) in smokers improves conduit artery vascular function, as evaluated by noninvasive flow-mediated dilation (FMD) (41, 43, 50) or by intra-arterial administration of endothelium-dependent vasodilators, such as ACh (17–19) and bradykinin (17). Furthermore, antioxidant-induced improvements in conduit artery vascular function in smokers are not observed when antioxidants are administered in conjunction with nitric oxide (NO) synthase (NOS) inhibition (31), suggesting that oxidative stress impairs conduit artery function by reducing NO bioavailability.

In addition to human conduit artery function, chronic cigarette smoking impairs function of the human microcirculation, such as the skin (9, 10, 14, 42). Given that microvascular dysfunction is a crucial step in the complications that lead to cardiovascular disease (1, 34, 38), exploring the mechanistic underpinnings of impaired microvascular function in smokers is important; however, few investigators have studied this issue. We (14) recently reported that young smokers have an impaired cutaneous vasodilatory response to administration of ACh compared with nonsmokers, which was partially due to attenuated NOS-dependent vasodilation. Given that oxidative stress reduces NO bioavailability in the conduit arteries of smokers (31), reducing oxidative stress may also improve NOS-dependent vasodilation in the cutaneous microcirculation of smokers, thereby enhancing vascular function. However, this has not been directly tested.

Using the above information as background, we hypothesized that tempol (a superoxide dismutase mimetic) would improve cutaneous vascular function through enhancing NOS-dependent vasodilation in young smokers. As a test of cutaneous vascular function, we evaluated cutaneous thermal hyperemia to local heating to 42°C at a rate of 0.1°C/sec. This test was selected as plateau vasodilation during local heating is predominantly (∼50–70%) mediated by NO (3, 5, 13, 20, 32, 39, 40, 49).

MATERIALS AND METHODS

Subjects.

This study was approved by the Institutional Review Board of The University of Oregon and conformed with guidelines set forth by the Declaration of Helsinki. Verbal and written informed consent were obtained from all subjects before their participation in the study. Smokers were defined as having smoked for at least 1 yr with an average daily cigarette consumption of ≥6 cigarettes/day. We recruited 10 healthy young (19–26 yr of age) smokers who had no history of hypertension, heart disease, diabetes, or autonomic disorders. This is important since advanced age (22, 26, 35, 40), hypertension (46), and disease status (e.g., chronic renal failure, postural tachycardia syndrome) (47, 48) are known to independently impair skin microvascular function. All subjects were not currently taking prescription medications. All subjects abstained from taking over-the-counter medications (including nonsteroidal anti-inflammatory agents and vitamins), alcohol, and caffeine for at least 24 h before the study. They also refrained from heavy exercise the night before the study and cigarette smoking for at least 12 h before the study to avoid any acute effects of cigarette smoking on skin blood flow regulation (9, 28, 52).

Instrumentation.

Upon arrival at the laboratory, subjects voided their bladder, and their body weight and height were measured. Subjects were placed in a semirecumbent position and instrumented with four microdialysis fibers (30-kDa cutoff, 10-mm membrane, MD2000, Bioanalytical Systems, West Lafayette, IN) on the ventral side of the forearm in the dermal layer of the skin. A 25-gauge needle was first inserted into the unanesthetized skin using aseptic techniques with at least 4.0 cm between each site. The entry and exit points were ∼2.5 cm apart. The microdialysis fiber was then threaded through the lumen of the needle, after which the needle was withdrawn leaving the fiber in place. Microdialysis fibers were secured with tape. Lactated Ringer solution was perfused through each microdialysis fiber at a rate of 2.0 μl/min (CMA 1025 microdialysis pump, CMA Microdialysis, Kista, Sweden) until the start of drug infusions (see below).

Experimental protocol.

Once the trauma caused by microdialysis fiber placement had dissipated (∼60–90 min), the experimental protocol began. Microdialysis fibers were randomly assigned to receive 1) lactated Ringer solution (control), 2) 10 μM 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol, EMD Millipore Chemicals, Billerica, MA) to reduce superoxide (O₂^·−), 3) 10 mM N^ω-nitro-l-arginine (l-NNA; Sigma-Aldrich, St. Louis, MO) to nonselectively inhibit NOS and thus NO production, and 4) 10 μM tempol plus 10 mM l-NNA. Drug concentrations were selected as the minimum dose required for maximal effects, as reported in previous studies (36, 37). All pharmacological agents were dissolved in lactated Ringer solution. All drugs were infused continuously at a rate of 2.0 μl/min (CMA 102 Microdialysis Pump, CMA Microdialysis) until the end of local heating to 42°C. To ensure adequate drug effects, all pharmacological agents were perfused for at least 75 min before the start of local heating.

After 75+ min of drug infusion, baseline was recorded for at least 10 min while skin temperature was held constant at 33°C (Skin Heater/Temperature Monitor SHO2, Moor Instruments, Devon, UK). Thereafter, local heating of the skin to 42°C at a rate of 0.1°C/s was applied to all skin sites to induce cutaneous vasodilation. Once skin vasodilation reached a plateau (25–35 min after the initiation of heating), local skin temperature was further elevated to 44°C at a rate of 0.1°C/s with an administration of 56 mM sodium nitroprusside (SNP; Nitropress, Ciba Pharmaceuticals, East Hanover, NJ) at a rate of 2.0 μl/min to achieve maximal vasodilation.

Measurements.

Arterial blood pressure was measured via automated brachial oscillation (Dinamap ProCare 100, GE Medical Systems, Tampa, FL) throughout the protocol. Mean arterial blood pressure (MAP) was calculated as diastolic arterial blood pressure plus one-third pulse pressure. To obtain an index of skin blood flow, cutaneous red blood cell flux was measured with a single-point laser-Doppler flowmetry probe (MoorLab; Moor Instruments) seated in the center of the local heater over each microdialysis fiber. Cutaneous vascular conductance (CVC) was evaluated as cutaneous red blood cell flux divided by MAP. All CVC data were expressed as percentages of maximal CVC to minimize the effect of site-to-site heterogeneity in the level of skin blood flow (38). Data were recorded and stored on a computer using Windaq data-acquisition software (Dataq Instruments, Akron, OH). Figure 1 shows the CVC response to local heating, averaged across all subjects, which was characterized as follows. Baseline CVC was determined by taking an average CVC at least over 3 min before heating. Upon the initiation of local heating, CVC rapidly increased and exhibited an initial peak. After a brief nadir, CVC then gradually increased and reached a stable plateau. The initial peak and nadir CVC were determined by taking averaged CVC over 30 s, and the plateau and maximal CVC were determined from averaged CVC over at least 2 min. We evaluated the difference in plateau CVC between tempol and tempol plus l-NNA sites as an index of NOS-dependent vasodilation with tempol. Similarly, the difference in plateau CVC between Ringer solution and l-NNA sites was evaluated as an index of NOS-dependent vasodilation without tempol.

Fig. 1.

Averaged time-course changes in cutaneous vascular conductance during local heating. Baseline, initial peak, nadir, and plateau cutaneous vascular conductance obtained from individuals were averaged and compared across the four drug sites. Time 0 indicates the initiation of local heating. Tempol, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, a superoxide dismutase mimetic; l-NNA, N^G-nitro-l-arginine (a nonspecific nitric oxide synthase inhibitor).

Statistical analyses.

Two-way repeated-measures ANOVA was conducted with factors of drug (Ringer solution, tempol, l-NNA, and combination of tempol and l-NNA) and phase of response (baseline, initial peak, nadir, plateau, and maximal periods) for absolute (mV/MAP × 100) and relative (%maximum) CVC. We used two-way ANOVA rather than one-way ANOVA to consider a potential interaction between drug and phase of response. When a significant main effect or interaction was detected, significant differences between paired variables across drug sites were determined by Tukey's honestly significant difference post hoc test. A two-tailed paired t-test was used to compare the difference in plateau CVC between tempol and tempol plus l-NNA sites with the difference in plateau CVC between Ringer solution and l-NNA sites. The level of significance was set at 0.05. Values are presented as means ± SE.

RESULTS

Characteristics of subjects.

Subjects were 22.5 ± 0.7 yr of age, with an average body mass index of 23.8 ± 0.8 kg/m². They had smoked for 6.3 ± 0.7 yr with an average daily consumption of 9.1 ± 0.7 cigarettes/day. Their systolic blood pressure, diastolic blood pressure, and MAP were 112.9 ± 2.7, 63.9 ± 1.8, and 80.2 ± 1.7 mmHg, respectively. Note that their body mass indexes and arterial blood pressures were within healthy ranges.

Cutaneous variables.

There was an interaction between drug and phase of response on CVC represented as both absolute and %maximum values (both P < 0.001). Plateau CVC at the tempol site was greater than that at the Ringer solution site (Fig. 1). Plateau CVC at the l-NNA site was reduced relative to the Ringer solution site (Fig. 1). Plateau CVC at the site that received combined tempol and l-NNA was lower compared with the Ringer solution site and was not different from the value at the l-NNA site (P = 0.978; Fig. 1). The difference in plateau CVC between the tempol and tempol plus l-NNA sites tended to be higher compared with the difference in plateau CVC between the Ringer solution and l-NNA sites (31.5 ± 4.1 vs. 19.2 ± 6.7%maximum, P = 0.163).

Baseline CVC at the Ringer solution site did not differ from that at the tempol (P = 0.999), l-NNA (P = 0.948), and tempol plus l-NNA (P = 0.999) sites (Fig. 1). Initial peak CVC at the Ringer solution site was not different from that at the tempol (P = 0.455), l-NNA (P = 0.095), and tempol plus l-NNA (P = 0.250) sites (Fig. 1). Absolute maximal CVC (mV/MAP × 100) at the Ringer solution site (246 ± 25) was not different from the tempol (314 ± 52, P = 0.180), l-NNA (277 ± 34, P = 0.790), and tempol plus l-NNA (292 ± 22, P = 0.514) sites. Based on these data, we calculated the minimum sample sizes required to produce a significant level of 0.05 with 80% power, which demonstrated we would need 18 subjects for the difference in baseline CVC between the Ringer solution and l-NNA sites to be significant, 28 subjects for the difference in initial peak CVC between the Ringer solution and tempol sites to be significant, 28 subjects for the difference in absolute maximal CVC between the Ringer solution and tempol sites to be significant, and 23 subjects for the difference in absolute maximal CVC between the Ringer solution and tempol plus l-NNA sites to be significant. Relative to the Ringer solution site, nadir CVC at the tempol site was higher, whereas that at the l-NNA and combination sites was lower (Fig. 1).

DISCUSSION

We are the first to investigate how tempol, a superoxide dismutase mimetic, affects the cutaneous vascular response to local heating in young smokers. We also used l-NNA (NOS inhibitor) to evaluate whether tempol-induced improvements in microvascular function were through improved NO bioavailability. Our main findings were that 1) tempol enhanced the plateau phase of cutaneous vasodilation to local heating to 42°C and 2) the plateau at the combination site (tempol plus l-NNA) was lower than at the Ringer solution site but was comparable to the l-NNA site. These results suggest that in young smokers, reducing oxidative stress in the microvasculature improves cutaneous thermal hyperemia to local heating through NO-dependent mechanisms.

Oxidative stress.

Accumulating evidence supports the concept that smokers have impaired cutaneous vascular function compared with nonsmokers (9, 10, 14, 42). In line with this, plateau CVC in the young smokers of the present study was attenuated compared with that in the young nonsmokers of previous studies in which the same heating protocol was used (Table 1) (3, 5, 16, 21, 33, 49, 54). In the present study, we found that tempol significantly improved plateau CVC compared with the Ringer solution site (Fig. 1), up to a similar level as the plateau in those same studies in nonsmokers (∼90%), suggesting that oxidative stress is a major factor contributing to the impaired cutaneous vascular function in young smokers.

Table 1.

Comparison of averaged plateau cutaneous vascular conductance during local heating at the control site between young smokers and nonsmokers

Author	Reference	Year	Plateau Cutaneous vascular conductance (%max)	Comment
Young smokers
Present study			77
Young nonsmokers
Hodges and Sparks	21	2014	86–90	At a rate of 0.5°C/10 s
Brunt and Minson	5	2012	84–88
Bruning et al.	3	2012	95
Greaney et al.	16	2012	93
Wong and Fieger	54	2010	92
Kellogg et al.	33	2008	85	Local heating to 41.5°C at a rate of 0.6°C/min
Stewart et al.	49	2008	91

All of the above studies used local heating to 42°C at a rate of 0.1°C/s unless otherwise indicated.

On the other hand, in healthy nonsmokers, antioxidants such as vitamin C do not affect vascular function, as evaluated by FMD or ACh-induced vasodilation in forearm conduit arteries (17, 19, 41, 43) as well as by the cutaneous vasodilatory response during whole body heating at rest (23, 25). More relevant to the present study, Medow et al. (36) showed in healthy young nonsmokers that tempol did not affect plateau CVC during the same local heating protocol as was used in the present study. The lack of an effect of antioxidants in healthy, nonsmoking subjects is not surprising, as healthy humans are expected not to have significant oxidative stress. Medow et al. (36) further showed that tempol restored plateau CVC when oxidative stress was induced in healthy nonsmokers by infusing angiotensin II, thus suggesting an antioxidative role of tempol during local heating. However, it should be considered that tempol may have nonantioxidative effects. For example, tempol-mediated opening of ATP-sensitive K⁺ channels has been reported in rats with systemic MAP changes (8), and opening of Ca²⁺-activated K⁺ (K_Ca) channels has been reported in rat mesenteric arterial smooth muscles (55). However, these effects are unlikely to have occurred in the present study, which focused on human skin blood flow regulation in smokers as no NO-independent tempol effects were observed, as discussed below.

NOS pathway.

Impaired NOS-dependent cutaneous vasodilation in smokers has been suggested by our previous study, which used ACh administration (14), and by the fact that, in the present study, NOS inhibition reduced plateau CVC during local heating to a lesser extent than what has previously been reported in healthy young nonsmokers (19 vs. 33–72%maximum) (3, 5, 13, 39, 40). Given that oxidative stress generally reduces NO bioavailability, it is plausible that in the present study, plateau CVC was improved with tempol administration by restoring NOS-dependent vasodilation. This notion is strongly supported by our observation that there was no difference in plateau CVC between l-NNA and tempol plus l-NNA sites (Fig. 1). Also, the difference in plateau CVC between the tempol and tempol plus l-NNA sites (an index of NOS-dependent vasodilation with tempol) tended to be higher compared with the difference in plateau CVC between the Ringer solution and l-NNA sites (an index of NOS-dependent vasodilation without tempol) (31.5 ± 4.1 vs. 19.2 ± 6.7%maximum, P = 0.163).

Possible mechanism(s) for how tempol improves NO bioavailability.

Cigarette smoking causes oxidative stress as a direct effect of the compounds within cigarette smoke itself (44). For example, the semiquinone radical in the cigarette tar yields O₂^·− (44). Additionally, NADPH oxidase, which produces O₂^·−, is directly activated by both nicotine, as shown in rat pial arterioles (12), and a stable thiol-reactive agent, as indicated in bovine, human, and rat pulmonary arteries (29). O₂^·− easily binds with NO to produce peroxynitrite, thus reducing NO bioavailability. Additionally, peroxynitrite depletes tetrahydrobiopterin, an essential cofactor for endothelial NOS (eNOS). This results in an increase in uncoupled eNOS, which then procures O₂^·− instead of NO (51), further reducing NO bioavailability. By removing O₂^·−, preventing it from binding with NO, and reducing uncoupled eNOS, tempol leads to higher NO bioavailability and thus improved plateau CVC. Moreover, the reaction of O₂^·− and tempol results in the production of H₂O₂, which may be another mechanism by which tempol improves plateau CVC. For example, scavenging of H₂O₂ with ebselen attenuates plateau CVC during local heating (36). Additionally, H₂O₂ can activate K_Ca channels, as shown in vascular smooth muscles of pig coronary arteries (2). Vasodilation via K_Ca channels contributes substantially to the plateau (5).

Limitations.

Tempol scavenges O₂^·− but not other ROS. Other ROS, such as H₂O₂ and hypochlorite may reduce NO bioavailability, as previously reported in porcine aortic endothelial cells (30), thus contributing to the attenuated plateau CVC during local heating in the skin of young smokers.

Only male subjects were included in this study. Thus, our conclusions cannot be applied to female subjects. Female sex hormones may be cardioprotective against the effects of chronic smoking, as reflected by the fact that the carotid and femoral artery wall thicknesses are greater (15) and conduit artery FMD is lower (6) in male smokers but not in female smokers compared with former or never smokers. Furthermore, female sex hormones enhance cutaneous thermal hyperemia to local heating (4, 7). Therefore, the effects of chronic cigarette smoking on cutaneous thermal hyperemia may be different between men and women and/or may be modulated by levels of female sex hormones. Further studies are warranted to address these issues.

Absolute maximal CVC at the tempol and tempol plus l-NNA sites tended to be higher relative to that at the Ringer solution site, although it was not significant due to the limited sample size. Reduced maximal cutaneous vasodilatory capacity has been reported in young (14) and older (10) smokers relative to nonsmoking counterparts. Our results suggest this may be due to oxidative stress, but further studies are required to flush that out.

For this study, we chose not to study a subset of nonsmokers, based on the number of studies showing no benefit of antioxidant administration on vascular responses in healthy, young nonsmokers. Although doing so would have allowed us to make comparisons between smokers and nonsmokers, we decided to specifically focus this study on investigating whether tempol would improve cutaneous vascular function in young smokers.

Perspectives.

Microvascular dysfunction may be a crucial step in the complications leading to cardiovascular disease and can be detected in the early stages of disease progression in the cutaneous circulation (24, 27, 38). The present study shows that impaired cutaneous microvascular function in young smokers is caused by oxidative stress in a similar fashion as is observed in aging (25). As such, chronic cigarette smoking has been suggested to cause a premature aging effect. Based on our results, we speculate that reducing oxidative stress in young smokers may potentially reduce the premature aging effect of chronic cigarette smoking on microvascular function, which, in turn, may prevent or delay smoking-related cardiovascular disease and mortality.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-081671. N. Fujii is the recipient of a research fellowship from the Uehara Memorial Foundation.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: N.F., V.E.B., and C.T.M. conception and design of research; N.F. and V.E.B. performed experiments; N.F. analyzed data; N.F., V.E.B., and C.T.M. interpreted results of experiments; N.F. prepared figures; N.F., V.E.B., and C.T.M. drafted manuscript; N.F., V.E.B., and C.T.M. edited and revised manuscript; N.F., V.E.B., and C.T.M. approved final version of manuscript.