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Inhaled carbon monoxide increases vasodilation in the microvascular circulation
Microvascular Research 123 (2019) 92–98
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Inhaled carbon monoxide increases vasodilation in the microvascular circulation Karalyn E. McRaea, Jessica Pudwellb, Nichole Petersona, Graeme N. Smitha,b, a b
T
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Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada Department of Obstetrics & Gynaecology, Queen's University, Kingston, Ontario, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Preeclampsia Carbon monoxide Microvasculature
Preeclampsia (PE) is characterized by systemic maternal endothelial dysfunction. Changes in endothelial reactivity have been reported before the onset of clinical signs of PE, and continuing into the post-partum period. Women who smoke during pregnancy have a 33% reduced risk of developing PE. This reduced risk is hypothesized to be, in part, attributed to carbon monoxide (CO), a by-product of cigarette combustion and a known endogenous vasodilator. Determining the vascular effects of CO in healthy women, may inform how CO can improve endothelial function and have promise as a novel therapeutic for PE. As part of a pilot study to determine the vascular effects of CO, the aim of this study was to measure microvascular vasodilation following low-dose CO inhalation. Non-pregnant women inhaled ambient room air or 250 ppm CO for 24 min during microvascular assessment using laser speckle contrast imaging. Changes in vascular flux were measured in the forearm before, during, and following a three-minute arterial occlusion. CO inhalation increased end-tidal breath CO (EtCO) (9.1 ± 1.9 vs. 1.8 ± 0.7 ppm, p < 0.05) and increased microvascular vasodilation, measured as difference of maximum level/resting level ratio (mean difference 0.476, 95% confidence interval (CI) = 0.149–0.802 vs. 0.118, 95% CI = −0.425–0.662, p < 0.05). Women who inhaled CO had a longer time to half recovery of endothelial function following arterial occlusion, compared to controls (hazard ratio 0.29, 95% CI = 0.10–0.91, p = 0.033). Inhalation of CO moderately increased EtCO and resulted in an increased microvascular response, suggesting that CO may have potential as a therapeutic for PE.
1. Introduction Preeclampsia (PE) is a multifactorial syndrome affecting 5–8% of pregnancies and is a leading cause of maternal and fetal morbidity and mortality (Roberts and Gammill, 2005). Central to PE is the development of systemic endothelial dysfunction (Khan et al., 2005). Endothelial dysfunction refers to a number of disrupted physiological processes, however the term is most often used to describe the inability of the endothelium to produce vasoactive factors, namely nitric oxide (NO) and prostacyclin (Drexler and Hornig, 1999). Nitric oxide is a key physiological vasodilator (Rudic et al., 1998) that acts on vascular smooth muscle cells by activation of soluble guanylyl cyclase (sGC) and subsequent production of cyclic guanosine monophosphate (cGMP)
(Matsubara et al., 2015). Endothelial dysfunction and decreased bioavailability of NO, is a shared characteristic of both PE and cardiovascular disease (CVD) (Ramsay et al., 2003). This relationship may explain why women with PE are at an increased lifetime risk of CVD compared to women with an uncomplicated obstetrical history (Smith et al., 2012; Smith et al., 2009; Brown et al., 2013). There is ongoing research to identify a reproducible, non-invasive method of measuring changes in endothelial function that can be used for screening and disease prediction. One such method being studied utilizes the microvessels of the skin. The cutaneous microvasculature is an accessible vascular bed that is representative of generalized endothelial function (Flammer et al., 2012; Holowatz et al., 2008). Abnormal endothelial function has been identified in the cutaneous
Abbreviations: Ach, acetylcholine; BMI, body mass index; BZ, biological zero; cGMP, cyclic guanosine monophosphate; CI, confidence interval; CO, carbon monoxide; COHb, carboxyhemoglobin; CPH, Cox proportional hazards; CVD, cardiovascular disease; Ca2+, calcium; EtCO, end-tidal carbon monoxide; HO, heme oxygenase; HR, hazard ratio; LSCI, laser speckle contrast imaging; ML, maximum level; NO, nitric oxide; NSAID, non-steroidal anti-inflammatory drugs; PE, preeclampsia; PORH, post-occlusive reactive hyperemia; PP, post-partum; ppm, parts per million; PU, perfusion unit; RL, resting level; ROI, region of interest; SD, standard deviation; sGC, soluble guanylyl cyclase; SNP, sodium nitroprusside; TH, time to half recovery ⁎ Corresponding author at: Department of Obstetrics & Gynaecology, Queen's University, Kingston Health Sciences Centre, 76 Stuart St., Kingston K7L 2V7, Canada. E-mail addresses: [email protected] (K.E. McRae), [email protected] (J. Pudwell), [email protected] (N. Peterson), [email protected] (G.N. Smith). https://doi.org/10.1016/j.mvr.2019.01.004 Received 16 October 2018; Received in revised form 10 January 2019; Accepted 10 January 2019 Available online 15 January 2019 0026-2862/ © 2019 Elsevier Inc. All rights reserved.
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microvasculature in PE (Khan et al., 2005; Murphy et al., 2014; Agatisa et al., 2004), obesity (de Jongh, 2004), diabetes (Khan et al., 2000), and CVD (Ijzerman et al., 2003). In PE, changes in endothelial function have been shown to develop early in pregnancy; microvascular dysfunction precedes macrovascular disease and the onset of clinical signs (Khan et al., 2005), and persists years into the post-partum (PP) period (Ramsay et al., 2003; Murphy et al., 2014; Agatisa et al., 2004). Although PE is more often described as impaired vasodilation in larger resistance vessels (Chambers et al., 2001; Savvidou et al., 2003), it has also been demonstrated that there is abnormal endothelium-dependent vasodilation in the microcirculation (Khan et al., 2005; Murphy et al., 2014; Blaauw et al., 2005). Studies measuring endothelial changes, triggered using perturbation by vasoactive agents, have been used to assess endothelial function both during pregnancy and in the early PP period. Murphy et al demonstrated that acetylcholine (ACh)-stimulated endothelium-dependent vasodilation was enhanced in the cutaneous microvasculature of women who had a previously PE pregnancy when measured 6 months PP compared to uncomplicated pregnancies (Murphy et al., 2014). In contrast, these women did not exhibit any changes in response to sodium nitroprusside (SNP), an endothelium-independent vasodilator (Murphy et al., 2014). It has been suggested that the enhanced ACh response occurring during PE may be due to a baseline vasoconstrictive state in the microvasculature (Blaauw et al., 2005), or a compensatory mechanism in response to impaired placental perfusion (Khan et al., 2005). It has also been suggested by Khan et al that the alterations in endothelial function may be due to underlying endothelial sensitization (Khan et al., 2005). Since there are measured increases in ACh (endothelium-dependent) but not SNP (endothelium-independent) responses, Khan et al suggests that this may be due, in part, to enhanced production of endothelium-derived vasoactive substances rather than increased responsiveness of the endothelium itself (Khan et al., 2005). Pre-existing endothelial sensitization may explain why endothelial dysfunction is prevalent in women who develop PE, however the relationship and mechanism have not yet been explored (Savvidou et al., 2003). Cigarette smoking has been shown to reduce the risk of developing PE by 33% in a dose-dependent manner (England and Zhang, 2007; England et al., 2002). This reduced incidence of PE is not seen in the population of smokeless tobacco users (Wikstrom et al., 2010), leading to the hypothesis that carbon monoxide (CO), one of the major combustible products of smoking, may be contributing to the reduced risk. CO is also produced endogenously by the catalytic degradation of heme by heme oxygenase (HO) (Maines, 1988). CO acts through a number of physiological pathways to control vascular tone (Coceani, 2000; Kozma et al., 1999; Leffler et al., 2011; Marks et al., 1991; Sammut et al., 1998), and activate anti-inflammatory (Cepinskas et al., 2007; Katada et al., 2010), anti-apoptotic (Bainbridge et al., 2006; Brouard et al., 2000), and cytoprotective (Clark, 2003) pathways. One of the reasons that CO is being explored as a potential therapeutic for PE is due to its ability to act via similar mechanisms as endothelium-derived NO. CO acts by activation of sGC (Utz and Ullrich, 1991; Coceani et al., 1996), smooth muscle cell large-conductance Ca2+-activated potassium channels (Clark, 2003; Wang et al., 1997; Jaggar, 2002; Dong et al., 2007; Wilkinson and Kemp, 2011), and inhibition of cytochrome p450 monooxygenase systems (Coceani et al., 1996; Coceani et al., 1988). As CO and NO appear to have parallel roles in the control of vascular tone, exogenous CO may be able to attenuate the endothelial dysfunction observed in women with PE. The aim of this study is to identify if exposure to low-dose inhaled CO can modify endothelial function in the peripheral cutaneous microvasculature. This study will utilize the innovative approach of laser speckle contrast imaging (LSCI) paired with post-occlusive reactive hyperemia (PORH) to quantify the spatiotemporal characteristics of the microvasculature during CO exposure in healthy non-pregnant women.
Table 1 Participant demographics and baseline characteristics.
Age (yrs) BMI (kg/m2) Ambient room temp (°C) Contraceptive use
Control group (air only) (n = 10)
Treatment group (CO) (n = 8)
p-Value
22.5 [22.0–25.0] 26.6 [21.9–29.7] 23.0 [22.5–23.1] 6 (60)
26.5 [22.8–31.0] 22.8 [20.2–25.8] 23.0 [22.8–23.3] 5 (62.5)
0.1091 0.2100 0.9226 1.00
Data presented as median [interquartile range] or n (%). Compared by the Mann-Whitney U test or Chi-Squared test.
2. Materials and methods 2.1. Ethics statement and subject recruitment All participants were recruited from the Kingston, Ontario community, and all work was completed at the Kingston Health Sciences Centre. This study was approved by the Queen's University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board (OBGY-261-14) and all participants provided written informed consent prior to participation. Non-smoking, non-pregnant women were recruited for microvascular assessment ± CO inhalation. Inhalation of CO was voluntary; neither participants or research staff were blinded to treatment group. Participants were eligible for the study if they were women, over 18 years of age, did not report any pre-existing cardiovascular, metabolic, or renal disease, and were never regular cigarette smokers. Demographic data was collected including age, body mass index (BMI), and contraceptive use (summarized in Table 1). On the day of microvascular assessment, participants were asked to refrain from sources of caffeine and medications (oral contraceptives and nonsteroidal anti-inflammatory drugs (NSAIDs)), to avoid potentially altering vascular measurements (Noguchi et al., 2015; Addor et al., 2008). 2.2. Carbon monoxide inhalation Medical grade CO (250 ppm, Praxair, Belleville, ON) was administered by a non-rebreather mask. Participants inhaled 250 ppm CO for 24 min, the duration of the microvascular assessment protocol. Microvascular reactivity was recorded for the duration of CO inhalation. The dose of 250 ppm CO was selected based on a review of pilot studies in the literature using 100–500 ppm CO for periods of up to 2 h (Mayr et al., 2005; Bathoorn et al., 2007). Levels of carboxyhemoglobin (COHb) were shown to be below that of typical smokers. The dose of 250 ppm CO has been used previously by our group, and the half-life in human subjects was determined to be 4.68 ± 1.83 h (Venditti and Smith, 2014). In that study, which assessed CO levels during two 1-h CO inhalations separated by a 4-h wash-out period, we reported no adverse health effects as blood COHb levels reached 6.00 ± 1.54% after the first hour, and 9.36 ± 2.14% following the second hour (Venditti and Smith, 2014). 2.3. Measurement of carbon monoxide in end-tidal breath samples End-tidal breath CO (EtCO) was measured using a piCO Smokerlyzer (Bedfont Scientific Ltd., Kent, England) immediately before and after the period of CO inhalation. The piCO Smokerlyzer displays all EtCO values within 30 s of a breath sample being blown into the digital apparatus. As reported in previous studies, a baseline EtCO of < 6 ppm, measured using noninvasive breath samples, is the standard cut-off for classification as a non-smoker (Middleton and Morice, 2000). Breath EtCO has been measured by our group in pregnant nonsmokers and smokers, and has been shown in the literature to correlate to reported cigarette use and COHb as measures of systemic CO (Venditti, 2009). 93
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Fig. 1. Representative images of microvascular flux acquisition using the moorFLP-2 LSCI. Images of the forearm at A) resting level, B) cuff inflation, C) biological zero during occlusion period, and D) reperfusion. Changes in mean microvascular flux (E) were graphed during each event (A–D) and graphed in relation to corresponding changes in occlusion cuff pressure (F).
2.4. Microvascular flux measurement by laser speckle contrast imaging
2.5. Post-occlusive reactive hyperemia
Laser speckle contrast imaging (LSCI) provides a real-time, noninvasive, assessment of vascular flux following perturbations to endothelial function (Briers et al., 2013; Mahe et al., 2012; Tew et al., 2011). Measurements of forearm microvascular flux were obtained using a MoorFLP-2 Laser Speckle Contrast Imager (Moor Instruments, Axminster, UK) (Fig. 1). Microvascular flux was expressed as arbitrary perfusion unit (PU). The laser imager was placed 20 cm above the forearm surface and images of forearm flux were recorded using Moor software at an acquisition rate of 25 frames/s. Flux was measured in a 1-in. diameter region of interest (ROI) on the right forearm, approximately 3 in. distal to the elbow, avoiding areas of excess hair and scarring. Measurements of microvascular flux in forearm regions has previously been validated (Addor et al., 2008; Yvonne-Tee et al., 2005), however there is no standardization for microvascular assessment. Participants were placed in a semi-supine position with their right arm supported on a hard matte black surface during all imaging. Participants were asked to remain still for the duration of the study to avoid causing movement artifacts. Room temperature was monitored for the duration of the study.
Study participants were divided into 2 groups, control and CO treatment. The experimental protocol for a PORH trial was 24 min in duration, consisting of a 1-min initiation period, a 10-min pre-occlusion period to obtain individual baseline resting levels, a 3-min arterial occlusion, and a 10-min post-occlusive period to measure hyperemia and return to resting levels. Each participant completed 2 PORH trials; trial 1 included inhalation of ambient air to quantify individual vascular responses, while trial 2 involved inhalation of ambient air (controls) or CO (treatment group). The second PORH trial was commenced only when the 10-min post-occlusion from trial 1 was complete, and it was confirmed that participants had returned to resting levels. PORH trials were completed consecutively with 1–5 min between trials. Participants in the CO treatment group inhaled 250 ppm CO for the duration of the 24-min PORH Trial 2. Arterial occlusion was initiated by inflating a MoorCMS-PRES automated occlusion cuff (Moor Instruments, Axeminster, UK) on the right upper arm to a suprasystolic pressure (200 millimeters of mercury (mmHg)) to occlude the brachial artery. The occlusion was maintained for a period of 3-mins, after which the cuff was rapidly deflated. MoorFLP-2 Review V5 PC software was used to analyze changes in flux (as measured in PU and represented colorimetrically (Fig. 1A–D)), and graphed as changes in flux (Fig. 1E) with respect to corresponding 94
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Table 2 Parameters calculated by moorFLP-2 software during PORH trials. Resting level (RL) Biological zero (BZ) Maximum level (ML) Time to half recovery (TH)
Average flux during pre-occlusion period Average flux during second half of pressure holding period Maximum flux value after cuff release Interval between cuff release and trace restoring half recovery back to RL
changes in occlusion pressure (Fig. 1F). Flux data collected during the PORH trials were analyzed using the manufacturer provided software for several parameters, summarized in Table 2. 2.6. Statistical analysis Demographic data are presented as median [interquartile range] or n (%) and were analyzed by the Mann-Whitney U test or Chi-Squared test, respectively. Carbon monoxide levels before and after CO inhalation (measured as EtCO) are presented as mean ± standard deviation (SD) and were analyzed by the Wilcoxon matched-pairs signed-rank test. Data from microvascular assessment is presented as the mean difference in maximum level (ML)/resting level (RL) ratio or maximum level (ML)/biological zero (BZ) between PORH trials (mean difference ± 95% confidence intervals (CI)), and analyzed by the MannWhitney U test. Since ratios were calculated in reference to individual resting levels, ML and RL measurements were not adjusted using BZ values. Data are presented as hazard ratios ± 95% CI. Comparison of time to half recovery (TH) between control and CO treatment groups was analyzed using a Cox Proportional Hazards (CPH) Model that adjusted for individual ML/RL ratios. Data were presented as a survival curve, depicting the percentage of participants in recovery vs. the time elapsed to reach a level of half recovery (the midpoint between max level and resting level microvascular response during the PORH postocclusion period). A survival curve was used in order to compare TH in control and CO groups, during both trial 1 (inhalation of air only) and trial 2 (inhalation of air vs. CO treatment). Analysis was completed using GraphPad Prism v6.0 or SPSS (CPH Model) with a significance of p < 0.05.
Fig. 2. EtCO levels following short exposure to inhaled CO. EtCO is elevated (9.1 ± 1.9 vs. 1.8 ± 0.7 ppm) following a short exposure (24 min) to 250 ppm inhaled CO (n = 8; data points for some baseline and post-CO measurement are superimposed on others). Analysis by Wilcoxon matched-pairs signed rank test with significance **p < 0.01.
CI = −0.425–0.662 p < 0.05) (Fig. 3A). CO had no statistically significant effect on the difference of the ML/BZ ratio (p > 0.05) (Fig. 3B). Using a CPH Model and adjusting for individual ML/RL ratios, TH was compared between control and experimental groups during both PORH trials (Fig. 4). No difference was observed during PORH trial 1 when both control and experimental groups were exposed to ambient room air (p > 0.05) (Fig. 4). During PORH trial 2 (corresponding to ambient room air vs. CO inhalation), TH was increased in the CO exposure group compared to controls (HR 0.29, 95% CI = 0.10–0.91, p = 0.033) (Fig. 4).
3. Results 3.1. Short exposure to inhaled carbon monoxide increases EtCO Participant demographics and baseline characteristics for control (n = 10) and CO treatment (n = 8) groups are summarized in Table 1. Age, BMI, and ambient room temperature were similar across all recruited participants (p > 0.05) (Table 1). Use of contraceptives was recorded as it has been previously shown to potentially alter microvascular flux (Cracowski et al., 2006). Biological CO levels were measured in participants immediately before and post-CO inhalation. CO was measured in breath samples as this method is rapid, noninvasive, and has been reported previously in non-pregnant and pregnant women, and following inhalation of CO (Venditti and Smith, 2014; Baum et al., 2000; Kreiser et al., 2004). After 24 mins of 250 ppm CO, EtCO was elevated (9.1 ± 1.9 ppm) compared to baseline levels (1.8 ± 0.7 ppm, p = 0.008) (Fig. 2).
4. Discussion A typical feature of PE is the development of widespread endothelial dysfunction (Agatisa et al., 2004; Chambers et al., 2001; Wang et al., 2004). Changes in endothelial reactivity and vessel resistance are associated with the development of maternal hypertension (Blaauw et al., 2005; Wang et al., 2004; Panza et al., 1990). It is well known that levels of CO are altered in PE (Baum et al., 2000; Kreiser et al., 2004; Ehsanipoor et al., 2013), and thus it has been suggested that CO, an endogenous vasodilator and anti-inflammatory molecule (Maines, 1988; Ryter et al., 2004; Vreman et al., 2000), may have potential as a novel therapeutic (Venditti and Smith, 2014; Bainbridge and Smith, 2005; Motterlini et al., 2005; Ryter and Choi, 2013; Ryter et al., 2006). This study aimed to investigate if increases in biological levels of CO modulate endothelial function in the microvascular circulation. Previous work from our group has examined inhalation of 250 ppm CO, determining that 1-h inhalation increase COHb to 6.00 ± 1.54% and a half-life of CO in non-pregnant women of 4.68 ± 1.83 h (Venditti and Smith, 2014). These levels of systemic CO are consistent with typical levels measured in smokers (Wald et al., 1981; Longo, 1970; Pojer et al., 1984), however it is plausible that much lower levels of CO may be sufficient to alter physiological processes and have beneficial effects in PE. The current study sought to measure changes in endothelial
3.2. Low dose carbon monoxide leads to measurable increases in microvascular dilation Microvascular reactivity data was expressed for each participant as the difference in the ratio of maximum level to resting level flux (ML/ RL) or maximum level to biological zero (ML/BZ) between PORH trials 1 and 2. This ratio is an indirect measure of microvascular vasodilation, accounting for inter-subject variability in resting flux levels. Inhalation of CO increased the difference of ML/RL ratio compared to controls (mean difference 0.476, 95% CI = 0.149–0.802 vs. 0.118, 95% 95
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surrogate endpoint in clinical studies of CVD (Addor et al., 2008). Four main factors are involved in the hyperemia response: metabolic vasodilators, endothelial vasodilators, myogenic response, and sensory nerves, as reported in a methodological review by Cracowski et al. (Cracowski et al., 2006). To our knowledge, this study is the first to use LSCI and PORH techniques to monitor changes in the microvascular circulation in response to CO exposure. We have demonstrated that CO inhalation results in microvascular vasodilation when measured as the difference in ML/RL ratios (Fig. 3). These findings are consistent with CO's known actions as a vasodilator, and the ability of CO to act by increasing cGMP (Coceani, 2000; Barbagallo et al., 2012) or increasing NO bioavailability (Thorup et al., 1999; Hartsfield, 2002). In this study, we also compared the time required for the endothelium to return to resting levels following perturbation by arterial occlusion. In order to account for individual differences in resting levels, this study was designed with two separate occlusion trials. We observed that in the second trial, control participants returned to resting level of flux faster than participants who inhaled low-dose CO during microvascular measurements (p < 0.05) (Fig. 4). Endothelial reactivity evaluates the response of the endothelium to metabolic and endothelial vasodilators (Cracowski et al., 2006). As such, the difference in response that we observed between the two trials, may be due to “priming” of the endothelium during the first trial. In the control group, TH is reduced in the second trial. We hypothesize that this may be due to the release of vasoactive factors and response of the endothelium that occurs in response to trial 1, leading to a faster response during trial 2, when these vasoactive factors may still be present in the microvascular environment. In the treatment group, the microvasculature is likely vasodilated by the presence of CO and, as such, would require an increased length of time to return to resting level of flux. Increasing our understanding of the association between biological levels of CO and microvascular response may provide a more thorough comprehension of how CO can be used to attenuate endothelial dysfunction in gestational complications, such as PE. A limitation of LSCI is that it is not a direct measure of blood flow. The premise of LSCI relies on the laser refraction from moving red blood cells to measure vascular flux, based on the velocity and density of red blood cell movement (Briers et al., 2013). Due to these assumptions, environmental factors such as room temperature, air flow, mental stress, and NSAID use, all affect flux calculations (Addor et al., 2008; Cracowski et al., 2006; Mahé et al., 2012). A strength of the current study is that all participant data were compared to individual resting levels, reducing the impact of confounding factors. Since the measurement of flux is made in arbitrary units and is not standardized, studies that measure microvascular flux can rarely be directly compared due to differences in methodology and equipment used. In this study we aimed to reduce as many external variables as possible. The ROI selected was 1-in. in diameter, larger than the typically used size of 10–100 mm, to reduce variability (Rousseau et al., 2011). Participants were also asked to refrain from caffeine and medications (oral contraceptives and NSAIDs) on the day of the study. All participants were of similar age, BMI, and no participants reported to be regular smokers or have any cardiovascular or metabolic disorders. Stage of menstrual cycle was not controlled in this study as it has previously been shown not to alter microvascular reactivity (Murphy et al., 2014). The exposure to inhaled CO in this study resulted in increased EtCO, albeit the CO levels were similar to light or occasional smokers. These minute increases in biological CO levels still had measurable physiological effects on the microvascular circulation. These results are promising, as they support the notion that low doses of CO may be beneficial in attenuating endothelial dysfunction and be a safe therapeutic for women with PE. Future studies are needed to elucidate the mechanistic role of CO on endothelial function, and to determine if it has similar effects in complicated pregnancies. Low-dose CO may help
Fig. 3. Ratio of maximum to resting flux levels in cutaneous microvasculature of the forearm. A) Microvascular vasodilation measured as the mean difference in ML/RL ratios, *p < 0.05. B) ML/BL ratios. Data graphed as mean difference ± 95% CI in control (n = 10) and CO treatment groups (n = 8), and analyzed by Mann-Whitney U test with significance of p < 0.05.
reactivity following a short exposure to CO. It has been proposed that measurements of endothelial function in the microvasculature may mirror events occurring in the systemic vasculature (Holowatz et al., 2008), and may help predict which women will develop PE. Previously, studies of endothelial function employed the use of Laser Doppler Flowmetry or Imaging. These techniques provided noninvasive methods to image the microvasculature, although only on a small scale. A strength of LSCI is the ability to image much larger regions, leading to less variability and more reproducible results (Rousseau et al., 2011). Post-occlusive reactive hyperemia measures flow-mediated dilation as alterations in microvascular flux before, during, and after an occlusion event. Maximal flux is observed immediately after pressure cuff release and measures the vasodilation caused by the ischemic event. Rather than measuring a single marker of endothelial function, PORH is an integrated measure of whole microvascular function (Cracowski et al., 2006). In previous studies, PORH in the forearm has been used as a 96
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Fig. 4. Time to endothelial recovery. Percent recovery between control and treatment groups during the room air inhalation trials (solid traces) in Trial 1. CO inhalation (red dotted trace) and controls (blue dotted trace) in Trial 2 (HR = 0.29, CI 0.10–0.91, p = 0.033). CPH model, adjusted for individual ML/RL ratios.
rescue endothelial dysfunction early in pregnancy in women who are either at high risk of developing, or have developed, PE. More research is needed on the safety of CO during pregnancy before this work can be continued in a population of pregnant women.
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Acknowledgements None. Funding This work was supported by an Ontario Graduate Scholarship awarded to KEM and a Canadian Institutes of Health Research (CIHR) Catalyst Grant (Grant #RN297868-371228) to GNS. The funding source had no role in study design; collection, analysis, and interpretation of data; in writing of the report; and decision to submit the article for publication. Declarations of interest None. Contributions KEM and GNS were responsible for concept and design of the study and experimental protocols. KEM carried out all experimental work and acquisition of data, with the help of NP. JP performed the statistical analysis on all data. KEM and GNS wrote the manuscript draft. Reviewed and editing by GNS. This manuscript has been read and approved by all authors. References Addor, G., Delachaux, A., Dischl, B., et al., 2008. A comparative study of reactive hyperemia in human forearm skin and muscle. Physiol. Res. 57 (5), 685. Agatisa, P.K., Ness, R.B., Roberts, J.M., Costantino, J.P., Kuller, L.H., McLaughlin, M.K., 2004. Impairment of endothelial function in women with a history of preeclampsia: an indicator of cardiovascular risk. Am. J. Physiol. Heart Circ. Physiol. 286 (4), H1389–H1393.
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Inhaled carbon monoxide increases vasodilation in the microvascular circulation Karalyn E. McRaea, Jessica Pudwellb, Nichole Petersona, Graeme N. Smitha,b, a b
T
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Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada Department of Obstetrics & Gynaecology, Queen's University, Kingston, Ontario, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Preeclampsia Carbon monoxide Microvasculature
Preeclampsia (PE) is characterized by systemic maternal endothelial dysfunction. Changes in endothelial reactivity have been reported before the onset of clinical signs of PE, and continuing into the post-partum period. Women who smoke during pregnancy have a 33% reduced risk of developing PE. This reduced risk is hypothesized to be, in part, attributed to carbon monoxide (CO), a by-product of cigarette combustion and a known endogenous vasodilator. Determining the vascular effects of CO in healthy women, may inform how CO can improve endothelial function and have promise as a novel therapeutic for PE. As part of a pilot study to determine the vascular effects of CO, the aim of this study was to measure microvascular vasodilation following low-dose CO inhalation. Non-pregnant women inhaled ambient room air or 250 ppm CO for 24 min during microvascular assessment using laser speckle contrast imaging. Changes in vascular flux were measured in the forearm before, during, and following a three-minute arterial occlusion. CO inhalation increased end-tidal breath CO (EtCO) (9.1 ± 1.9 vs. 1.8 ± 0.7 ppm, p < 0.05) and increased microvascular vasodilation, measured as difference of maximum level/resting level ratio (mean difference 0.476, 95% confidence interval (CI) = 0.149–0.802 vs. 0.118, 95% CI = −0.425–0.662, p < 0.05). Women who inhaled CO had a longer time to half recovery of endothelial function following arterial occlusion, compared to controls (hazard ratio 0.29, 95% CI = 0.10–0.91, p = 0.033). Inhalation of CO moderately increased EtCO and resulted in an increased microvascular response, suggesting that CO may have potential as a therapeutic for PE.
1. Introduction Preeclampsia (PE) is a multifactorial syndrome affecting 5–8% of pregnancies and is a leading cause of maternal and fetal morbidity and mortality (Roberts and Gammill, 2005). Central to PE is the development of systemic endothelial dysfunction (Khan et al., 2005). Endothelial dysfunction refers to a number of disrupted physiological processes, however the term is most often used to describe the inability of the endothelium to produce vasoactive factors, namely nitric oxide (NO) and prostacyclin (Drexler and Hornig, 1999). Nitric oxide is a key physiological vasodilator (Rudic et al., 1998) that acts on vascular smooth muscle cells by activation of soluble guanylyl cyclase (sGC) and subsequent production of cyclic guanosine monophosphate (cGMP)
(Matsubara et al., 2015). Endothelial dysfunction and decreased bioavailability of NO, is a shared characteristic of both PE and cardiovascular disease (CVD) (Ramsay et al., 2003). This relationship may explain why women with PE are at an increased lifetime risk of CVD compared to women with an uncomplicated obstetrical history (Smith et al., 2012; Smith et al., 2009; Brown et al., 2013). There is ongoing research to identify a reproducible, non-invasive method of measuring changes in endothelial function that can be used for screening and disease prediction. One such method being studied utilizes the microvessels of the skin. The cutaneous microvasculature is an accessible vascular bed that is representative of generalized endothelial function (Flammer et al., 2012; Holowatz et al., 2008). Abnormal endothelial function has been identified in the cutaneous
Abbreviations: Ach, acetylcholine; BMI, body mass index; BZ, biological zero; cGMP, cyclic guanosine monophosphate; CI, confidence interval; CO, carbon monoxide; COHb, carboxyhemoglobin; CPH, Cox proportional hazards; CVD, cardiovascular disease; Ca2+, calcium; EtCO, end-tidal carbon monoxide; HO, heme oxygenase; HR, hazard ratio; LSCI, laser speckle contrast imaging; ML, maximum level; NO, nitric oxide; NSAID, non-steroidal anti-inflammatory drugs; PE, preeclampsia; PORH, post-occlusive reactive hyperemia; PP, post-partum; ppm, parts per million; PU, perfusion unit; RL, resting level; ROI, region of interest; SD, standard deviation; sGC, soluble guanylyl cyclase; SNP, sodium nitroprusside; TH, time to half recovery ⁎ Corresponding author at: Department of Obstetrics & Gynaecology, Queen's University, Kingston Health Sciences Centre, 76 Stuart St., Kingston K7L 2V7, Canada. E-mail addresses: [email protected] (K.E. McRae), [email protected] (J. Pudwell), [email protected] (N. Peterson), [email protected] (G.N. Smith). https://doi.org/10.1016/j.mvr.2019.01.004 Received 16 October 2018; Received in revised form 10 January 2019; Accepted 10 January 2019 Available online 15 January 2019 0026-2862/ © 2019 Elsevier Inc. All rights reserved.
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microvasculature in PE (Khan et al., 2005; Murphy et al., 2014; Agatisa et al., 2004), obesity (de Jongh, 2004), diabetes (Khan et al., 2000), and CVD (Ijzerman et al., 2003). In PE, changes in endothelial function have been shown to develop early in pregnancy; microvascular dysfunction precedes macrovascular disease and the onset of clinical signs (Khan et al., 2005), and persists years into the post-partum (PP) period (Ramsay et al., 2003; Murphy et al., 2014; Agatisa et al., 2004). Although PE is more often described as impaired vasodilation in larger resistance vessels (Chambers et al., 2001; Savvidou et al., 2003), it has also been demonstrated that there is abnormal endothelium-dependent vasodilation in the microcirculation (Khan et al., 2005; Murphy et al., 2014; Blaauw et al., 2005). Studies measuring endothelial changes, triggered using perturbation by vasoactive agents, have been used to assess endothelial function both during pregnancy and in the early PP period. Murphy et al demonstrated that acetylcholine (ACh)-stimulated endothelium-dependent vasodilation was enhanced in the cutaneous microvasculature of women who had a previously PE pregnancy when measured 6 months PP compared to uncomplicated pregnancies (Murphy et al., 2014). In contrast, these women did not exhibit any changes in response to sodium nitroprusside (SNP), an endothelium-independent vasodilator (Murphy et al., 2014). It has been suggested that the enhanced ACh response occurring during PE may be due to a baseline vasoconstrictive state in the microvasculature (Blaauw et al., 2005), or a compensatory mechanism in response to impaired placental perfusion (Khan et al., 2005). It has also been suggested by Khan et al that the alterations in endothelial function may be due to underlying endothelial sensitization (Khan et al., 2005). Since there are measured increases in ACh (endothelium-dependent) but not SNP (endothelium-independent) responses, Khan et al suggests that this may be due, in part, to enhanced production of endothelium-derived vasoactive substances rather than increased responsiveness of the endothelium itself (Khan et al., 2005). Pre-existing endothelial sensitization may explain why endothelial dysfunction is prevalent in women who develop PE, however the relationship and mechanism have not yet been explored (Savvidou et al., 2003). Cigarette smoking has been shown to reduce the risk of developing PE by 33% in a dose-dependent manner (England and Zhang, 2007; England et al., 2002). This reduced incidence of PE is not seen in the population of smokeless tobacco users (Wikstrom et al., 2010), leading to the hypothesis that carbon monoxide (CO), one of the major combustible products of smoking, may be contributing to the reduced risk. CO is also produced endogenously by the catalytic degradation of heme by heme oxygenase (HO) (Maines, 1988). CO acts through a number of physiological pathways to control vascular tone (Coceani, 2000; Kozma et al., 1999; Leffler et al., 2011; Marks et al., 1991; Sammut et al., 1998), and activate anti-inflammatory (Cepinskas et al., 2007; Katada et al., 2010), anti-apoptotic (Bainbridge et al., 2006; Brouard et al., 2000), and cytoprotective (Clark, 2003) pathways. One of the reasons that CO is being explored as a potential therapeutic for PE is due to its ability to act via similar mechanisms as endothelium-derived NO. CO acts by activation of sGC (Utz and Ullrich, 1991; Coceani et al., 1996), smooth muscle cell large-conductance Ca2+-activated potassium channels (Clark, 2003; Wang et al., 1997; Jaggar, 2002; Dong et al., 2007; Wilkinson and Kemp, 2011), and inhibition of cytochrome p450 monooxygenase systems (Coceani et al., 1996; Coceani et al., 1988). As CO and NO appear to have parallel roles in the control of vascular tone, exogenous CO may be able to attenuate the endothelial dysfunction observed in women with PE. The aim of this study is to identify if exposure to low-dose inhaled CO can modify endothelial function in the peripheral cutaneous microvasculature. This study will utilize the innovative approach of laser speckle contrast imaging (LSCI) paired with post-occlusive reactive hyperemia (PORH) to quantify the spatiotemporal characteristics of the microvasculature during CO exposure in healthy non-pregnant women.
Table 1 Participant demographics and baseline characteristics.
Age (yrs) BMI (kg/m2) Ambient room temp (°C) Contraceptive use
Control group (air only) (n = 10)
Treatment group (CO) (n = 8)
p-Value
22.5 [22.0–25.0] 26.6 [21.9–29.7] 23.0 [22.5–23.1] 6 (60)
26.5 [22.8–31.0] 22.8 [20.2–25.8] 23.0 [22.8–23.3] 5 (62.5)
0.1091 0.2100 0.9226 1.00
Data presented as median [interquartile range] or n (%). Compared by the Mann-Whitney U test or Chi-Squared test.
2. Materials and methods 2.1. Ethics statement and subject recruitment All participants were recruited from the Kingston, Ontario community, and all work was completed at the Kingston Health Sciences Centre. This study was approved by the Queen's University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board (OBGY-261-14) and all participants provided written informed consent prior to participation. Non-smoking, non-pregnant women were recruited for microvascular assessment ± CO inhalation. Inhalation of CO was voluntary; neither participants or research staff were blinded to treatment group. Participants were eligible for the study if they were women, over 18 years of age, did not report any pre-existing cardiovascular, metabolic, or renal disease, and were never regular cigarette smokers. Demographic data was collected including age, body mass index (BMI), and contraceptive use (summarized in Table 1). On the day of microvascular assessment, participants were asked to refrain from sources of caffeine and medications (oral contraceptives and nonsteroidal anti-inflammatory drugs (NSAIDs)), to avoid potentially altering vascular measurements (Noguchi et al., 2015; Addor et al., 2008). 2.2. Carbon monoxide inhalation Medical grade CO (250 ppm, Praxair, Belleville, ON) was administered by a non-rebreather mask. Participants inhaled 250 ppm CO for 24 min, the duration of the microvascular assessment protocol. Microvascular reactivity was recorded for the duration of CO inhalation. The dose of 250 ppm CO was selected based on a review of pilot studies in the literature using 100–500 ppm CO for periods of up to 2 h (Mayr et al., 2005; Bathoorn et al., 2007). Levels of carboxyhemoglobin (COHb) were shown to be below that of typical smokers. The dose of 250 ppm CO has been used previously by our group, and the half-life in human subjects was determined to be 4.68 ± 1.83 h (Venditti and Smith, 2014). In that study, which assessed CO levels during two 1-h CO inhalations separated by a 4-h wash-out period, we reported no adverse health effects as blood COHb levels reached 6.00 ± 1.54% after the first hour, and 9.36 ± 2.14% following the second hour (Venditti and Smith, 2014). 2.3. Measurement of carbon monoxide in end-tidal breath samples End-tidal breath CO (EtCO) was measured using a piCO Smokerlyzer (Bedfont Scientific Ltd., Kent, England) immediately before and after the period of CO inhalation. The piCO Smokerlyzer displays all EtCO values within 30 s of a breath sample being blown into the digital apparatus. As reported in previous studies, a baseline EtCO of < 6 ppm, measured using noninvasive breath samples, is the standard cut-off for classification as a non-smoker (Middleton and Morice, 2000). Breath EtCO has been measured by our group in pregnant nonsmokers and smokers, and has been shown in the literature to correlate to reported cigarette use and COHb as measures of systemic CO (Venditti, 2009). 93
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Fig. 1. Representative images of microvascular flux acquisition using the moorFLP-2 LSCI. Images of the forearm at A) resting level, B) cuff inflation, C) biological zero during occlusion period, and D) reperfusion. Changes in mean microvascular flux (E) were graphed during each event (A–D) and graphed in relation to corresponding changes in occlusion cuff pressure (F).
2.4. Microvascular flux measurement by laser speckle contrast imaging
2.5. Post-occlusive reactive hyperemia
Laser speckle contrast imaging (LSCI) provides a real-time, noninvasive, assessment of vascular flux following perturbations to endothelial function (Briers et al., 2013; Mahe et al., 2012; Tew et al., 2011). Measurements of forearm microvascular flux were obtained using a MoorFLP-2 Laser Speckle Contrast Imager (Moor Instruments, Axminster, UK) (Fig. 1). Microvascular flux was expressed as arbitrary perfusion unit (PU). The laser imager was placed 20 cm above the forearm surface and images of forearm flux were recorded using Moor software at an acquisition rate of 25 frames/s. Flux was measured in a 1-in. diameter region of interest (ROI) on the right forearm, approximately 3 in. distal to the elbow, avoiding areas of excess hair and scarring. Measurements of microvascular flux in forearm regions has previously been validated (Addor et al., 2008; Yvonne-Tee et al., 2005), however there is no standardization for microvascular assessment. Participants were placed in a semi-supine position with their right arm supported on a hard matte black surface during all imaging. Participants were asked to remain still for the duration of the study to avoid causing movement artifacts. Room temperature was monitored for the duration of the study.
Study participants were divided into 2 groups, control and CO treatment. The experimental protocol for a PORH trial was 24 min in duration, consisting of a 1-min initiation period, a 10-min pre-occlusion period to obtain individual baseline resting levels, a 3-min arterial occlusion, and a 10-min post-occlusive period to measure hyperemia and return to resting levels. Each participant completed 2 PORH trials; trial 1 included inhalation of ambient air to quantify individual vascular responses, while trial 2 involved inhalation of ambient air (controls) or CO (treatment group). The second PORH trial was commenced only when the 10-min post-occlusion from trial 1 was complete, and it was confirmed that participants had returned to resting levels. PORH trials were completed consecutively with 1–5 min between trials. Participants in the CO treatment group inhaled 250 ppm CO for the duration of the 24-min PORH Trial 2. Arterial occlusion was initiated by inflating a MoorCMS-PRES automated occlusion cuff (Moor Instruments, Axeminster, UK) on the right upper arm to a suprasystolic pressure (200 millimeters of mercury (mmHg)) to occlude the brachial artery. The occlusion was maintained for a period of 3-mins, after which the cuff was rapidly deflated. MoorFLP-2 Review V5 PC software was used to analyze changes in flux (as measured in PU and represented colorimetrically (Fig. 1A–D)), and graphed as changes in flux (Fig. 1E) with respect to corresponding 94
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Table 2 Parameters calculated by moorFLP-2 software during PORH trials. Resting level (RL) Biological zero (BZ) Maximum level (ML) Time to half recovery (TH)
Average flux during pre-occlusion period Average flux during second half of pressure holding period Maximum flux value after cuff release Interval between cuff release and trace restoring half recovery back to RL
changes in occlusion pressure (Fig. 1F). Flux data collected during the PORH trials were analyzed using the manufacturer provided software for several parameters, summarized in Table 2. 2.6. Statistical analysis Demographic data are presented as median [interquartile range] or n (%) and were analyzed by the Mann-Whitney U test or Chi-Squared test, respectively. Carbon monoxide levels before and after CO inhalation (measured as EtCO) are presented as mean ± standard deviation (SD) and were analyzed by the Wilcoxon matched-pairs signed-rank test. Data from microvascular assessment is presented as the mean difference in maximum level (ML)/resting level (RL) ratio or maximum level (ML)/biological zero (BZ) between PORH trials (mean difference ± 95% confidence intervals (CI)), and analyzed by the MannWhitney U test. Since ratios were calculated in reference to individual resting levels, ML and RL measurements were not adjusted using BZ values. Data are presented as hazard ratios ± 95% CI. Comparison of time to half recovery (TH) between control and CO treatment groups was analyzed using a Cox Proportional Hazards (CPH) Model that adjusted for individual ML/RL ratios. Data were presented as a survival curve, depicting the percentage of participants in recovery vs. the time elapsed to reach a level of half recovery (the midpoint between max level and resting level microvascular response during the PORH postocclusion period). A survival curve was used in order to compare TH in control and CO groups, during both trial 1 (inhalation of air only) and trial 2 (inhalation of air vs. CO treatment). Analysis was completed using GraphPad Prism v6.0 or SPSS (CPH Model) with a significance of p < 0.05.
Fig. 2. EtCO levels following short exposure to inhaled CO. EtCO is elevated (9.1 ± 1.9 vs. 1.8 ± 0.7 ppm) following a short exposure (24 min) to 250 ppm inhaled CO (n = 8; data points for some baseline and post-CO measurement are superimposed on others). Analysis by Wilcoxon matched-pairs signed rank test with significance **p < 0.01.
CI = −0.425–0.662 p < 0.05) (Fig. 3A). CO had no statistically significant effect on the difference of the ML/BZ ratio (p > 0.05) (Fig. 3B). Using a CPH Model and adjusting for individual ML/RL ratios, TH was compared between control and experimental groups during both PORH trials (Fig. 4). No difference was observed during PORH trial 1 when both control and experimental groups were exposed to ambient room air (p > 0.05) (Fig. 4). During PORH trial 2 (corresponding to ambient room air vs. CO inhalation), TH was increased in the CO exposure group compared to controls (HR 0.29, 95% CI = 0.10–0.91, p = 0.033) (Fig. 4).
3. Results 3.1. Short exposure to inhaled carbon monoxide increases EtCO Participant demographics and baseline characteristics for control (n = 10) and CO treatment (n = 8) groups are summarized in Table 1. Age, BMI, and ambient room temperature were similar across all recruited participants (p > 0.05) (Table 1). Use of contraceptives was recorded as it has been previously shown to potentially alter microvascular flux (Cracowski et al., 2006). Biological CO levels were measured in participants immediately before and post-CO inhalation. CO was measured in breath samples as this method is rapid, noninvasive, and has been reported previously in non-pregnant and pregnant women, and following inhalation of CO (Venditti and Smith, 2014; Baum et al., 2000; Kreiser et al., 2004). After 24 mins of 250 ppm CO, EtCO was elevated (9.1 ± 1.9 ppm) compared to baseline levels (1.8 ± 0.7 ppm, p = 0.008) (Fig. 2).
4. Discussion A typical feature of PE is the development of widespread endothelial dysfunction (Agatisa et al., 2004; Chambers et al., 2001; Wang et al., 2004). Changes in endothelial reactivity and vessel resistance are associated with the development of maternal hypertension (Blaauw et al., 2005; Wang et al., 2004; Panza et al., 1990). It is well known that levels of CO are altered in PE (Baum et al., 2000; Kreiser et al., 2004; Ehsanipoor et al., 2013), and thus it has been suggested that CO, an endogenous vasodilator and anti-inflammatory molecule (Maines, 1988; Ryter et al., 2004; Vreman et al., 2000), may have potential as a novel therapeutic (Venditti and Smith, 2014; Bainbridge and Smith, 2005; Motterlini et al., 2005; Ryter and Choi, 2013; Ryter et al., 2006). This study aimed to investigate if increases in biological levels of CO modulate endothelial function in the microvascular circulation. Previous work from our group has examined inhalation of 250 ppm CO, determining that 1-h inhalation increase COHb to 6.00 ± 1.54% and a half-life of CO in non-pregnant women of 4.68 ± 1.83 h (Venditti and Smith, 2014). These levels of systemic CO are consistent with typical levels measured in smokers (Wald et al., 1981; Longo, 1970; Pojer et al., 1984), however it is plausible that much lower levels of CO may be sufficient to alter physiological processes and have beneficial effects in PE. The current study sought to measure changes in endothelial
3.2. Low dose carbon monoxide leads to measurable increases in microvascular dilation Microvascular reactivity data was expressed for each participant as the difference in the ratio of maximum level to resting level flux (ML/ RL) or maximum level to biological zero (ML/BZ) between PORH trials 1 and 2. This ratio is an indirect measure of microvascular vasodilation, accounting for inter-subject variability in resting flux levels. Inhalation of CO increased the difference of ML/RL ratio compared to controls (mean difference 0.476, 95% CI = 0.149–0.802 vs. 0.118, 95% 95
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surrogate endpoint in clinical studies of CVD (Addor et al., 2008). Four main factors are involved in the hyperemia response: metabolic vasodilators, endothelial vasodilators, myogenic response, and sensory nerves, as reported in a methodological review by Cracowski et al. (Cracowski et al., 2006). To our knowledge, this study is the first to use LSCI and PORH techniques to monitor changes in the microvascular circulation in response to CO exposure. We have demonstrated that CO inhalation results in microvascular vasodilation when measured as the difference in ML/RL ratios (Fig. 3). These findings are consistent with CO's known actions as a vasodilator, and the ability of CO to act by increasing cGMP (Coceani, 2000; Barbagallo et al., 2012) or increasing NO bioavailability (Thorup et al., 1999; Hartsfield, 2002). In this study, we also compared the time required for the endothelium to return to resting levels following perturbation by arterial occlusion. In order to account for individual differences in resting levels, this study was designed with two separate occlusion trials. We observed that in the second trial, control participants returned to resting level of flux faster than participants who inhaled low-dose CO during microvascular measurements (p < 0.05) (Fig. 4). Endothelial reactivity evaluates the response of the endothelium to metabolic and endothelial vasodilators (Cracowski et al., 2006). As such, the difference in response that we observed between the two trials, may be due to “priming” of the endothelium during the first trial. In the control group, TH is reduced in the second trial. We hypothesize that this may be due to the release of vasoactive factors and response of the endothelium that occurs in response to trial 1, leading to a faster response during trial 2, when these vasoactive factors may still be present in the microvascular environment. In the treatment group, the microvasculature is likely vasodilated by the presence of CO and, as such, would require an increased length of time to return to resting level of flux. Increasing our understanding of the association between biological levels of CO and microvascular response may provide a more thorough comprehension of how CO can be used to attenuate endothelial dysfunction in gestational complications, such as PE. A limitation of LSCI is that it is not a direct measure of blood flow. The premise of LSCI relies on the laser refraction from moving red blood cells to measure vascular flux, based on the velocity and density of red blood cell movement (Briers et al., 2013). Due to these assumptions, environmental factors such as room temperature, air flow, mental stress, and NSAID use, all affect flux calculations (Addor et al., 2008; Cracowski et al., 2006; Mahé et al., 2012). A strength of the current study is that all participant data were compared to individual resting levels, reducing the impact of confounding factors. Since the measurement of flux is made in arbitrary units and is not standardized, studies that measure microvascular flux can rarely be directly compared due to differences in methodology and equipment used. In this study we aimed to reduce as many external variables as possible. The ROI selected was 1-in. in diameter, larger than the typically used size of 10–100 mm, to reduce variability (Rousseau et al., 2011). Participants were also asked to refrain from caffeine and medications (oral contraceptives and NSAIDs) on the day of the study. All participants were of similar age, BMI, and no participants reported to be regular smokers or have any cardiovascular or metabolic disorders. Stage of menstrual cycle was not controlled in this study as it has previously been shown not to alter microvascular reactivity (Murphy et al., 2014). The exposure to inhaled CO in this study resulted in increased EtCO, albeit the CO levels were similar to light or occasional smokers. These minute increases in biological CO levels still had measurable physiological effects on the microvascular circulation. These results are promising, as they support the notion that low doses of CO may be beneficial in attenuating endothelial dysfunction and be a safe therapeutic for women with PE. Future studies are needed to elucidate the mechanistic role of CO on endothelial function, and to determine if it has similar effects in complicated pregnancies. Low-dose CO may help
Fig. 3. Ratio of maximum to resting flux levels in cutaneous microvasculature of the forearm. A) Microvascular vasodilation measured as the mean difference in ML/RL ratios, *p < 0.05. B) ML/BL ratios. Data graphed as mean difference ± 95% CI in control (n = 10) and CO treatment groups (n = 8), and analyzed by Mann-Whitney U test with significance of p < 0.05.
reactivity following a short exposure to CO. It has been proposed that measurements of endothelial function in the microvasculature may mirror events occurring in the systemic vasculature (Holowatz et al., 2008), and may help predict which women will develop PE. Previously, studies of endothelial function employed the use of Laser Doppler Flowmetry or Imaging. These techniques provided noninvasive methods to image the microvasculature, although only on a small scale. A strength of LSCI is the ability to image much larger regions, leading to less variability and more reproducible results (Rousseau et al., 2011). Post-occlusive reactive hyperemia measures flow-mediated dilation as alterations in microvascular flux before, during, and after an occlusion event. Maximal flux is observed immediately after pressure cuff release and measures the vasodilation caused by the ischemic event. Rather than measuring a single marker of endothelial function, PORH is an integrated measure of whole microvascular function (Cracowski et al., 2006). In previous studies, PORH in the forearm has been used as a 96
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Fig. 4. Time to endothelial recovery. Percent recovery between control and treatment groups during the room air inhalation trials (solid traces) in Trial 1. CO inhalation (red dotted trace) and controls (blue dotted trace) in Trial 2 (HR = 0.29, CI 0.10–0.91, p = 0.033). CPH model, adjusted for individual ML/RL ratios.
rescue endothelial dysfunction early in pregnancy in women who are either at high risk of developing, or have developed, PE. More research is needed on the safety of CO during pregnancy before this work can be continued in a population of pregnant women.
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Acknowledgements None. Funding This work was supported by an Ontario Graduate Scholarship awarded to KEM and a Canadian Institutes of Health Research (CIHR) Catalyst Grant (Grant #RN297868-371228) to GNS. The funding source had no role in study design; collection, analysis, and interpretation of data; in writing of the report; and decision to submit the article for publication. Declarations of interest None. Contributions KEM and GNS were responsible for concept and design of the study and experimental protocols. KEM carried out all experimental work and acquisition of data, with the help of NP. JP performed the statistical analysis on all data. KEM and GNS wrote the manuscript draft. Reviewed and editing by GNS. This manuscript has been read and approved by all authors. References Addor, G., Delachaux, A., Dischl, B., et al., 2008. A comparative study of reactive hyperemia in human forearm skin and muscle. Physiol. Res. 57 (5), 685. Agatisa, P.K., Ness, R.B., Roberts, J.M., Costantino, J.P., Kuller, L.H., McLaughlin, M.K., 2004. Impairment of endothelial function in women with a history of preeclampsia: an indicator of cardiovascular risk. Am. J. Physiol. Heart Circ. Physiol. 286 (4), H1389–H1393.
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