The impact of acute mental stress on brachial artery flow-mediated dilation in women diagnosed with depression

International Journal of Psychophysiology 135 (2019) 113–120

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The impact of acute mental stress on brachial artery flow-mediated dilation in women diagnosed with depression

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Katrina A. D'Urzoa, Cherie L. La Rocqueb, Jennifer S. Williamsa, Troy J.R. Stucklessa, ⁎ Trevor J. Kinga, Meghan D. Plotnicka, Brendon J. Gurda, Kate L. Harknessb, Kyra E. Pykea, a b

School of Kinesiology and Health Studies, 28 Division Street, Queen's University, Kingston K7L 3N6, Ontario, Canada Department of Psychology, 62 Arch Street, Queen's University, Kingston K7L 3N6, Ontario, Canada

ARTICLE INFO

ABSTRACT

Keywords: Endothelial function FMD Reactive hyperemia Trier social stress test Cortisol

Endothelial function, assessed by flow-mediated dilation (FMD), may be transiently attenuated in healthy adults following acute mental stress. However, the impact of acute mental stress on endothelial function in the context of clinical depression is unknown. This study examined the impact of acute mental stress on FMD in women with a diagnosis of a depressive disorder. Forty-three otherwise healthy women (33 ± 14 years) participated. Brachial artery diameter and blood velocity were assessed with ultrasound. FMD was assessed immediately prior to and 15 min following the Trier Social Stress Test (TSST). The FMD protocol included 5 min of forearm cuff occlusion (pressure = 250 mm Hg), followed by release. Shear stress was estimated by calculating shear rate (SR = brachial artery blood velocity/diameter). Stress reactivity was assessed via changes in mean arterial pressure (MAP), heart rate (HR) and salivary cortisol. Results are mean ± SD. A significant stress response was elicited by the TSST [MAP, HR and salivary cortisol increased (p < 0.05)]. Neither the SR stimulus nor FMD response differed pre-versus post-stress (p = 0.124 and p = 0.641, respectively). There was a modest negative correlation between cortisol reactivity and change in FMD from pre- to post-stress (R = −0.392, p = 0.011). To conclude, acute mental stress did not consistently impair endothelial function in women diagnosed with a depressive disorder; however, higher cortisol reactivity may increase the likelihood of post-stress endothelial dysfunction. Further research is required to better understand the factors influencing the relationship between acute mental stress, cortisol and endothelial function in women with depression.

1. Introduction Transient impairments in endothelial function, as assessed by flowmediated dilation (FMD), have been reported in healthy adults following acute mental stress (Broadley et al., 2005; Ghiadoni et al., 2000; Spieker et al., 2002). This immediate impact of stress on endothelial function may play a role in the chronic interaction between stress and cardiovascular health (Richardson et al., 2012). For example, during a stressful event, it is possible that endothelial dysfunction contributes to the development of acute myocardial ischemia, and repeated temporary dysfunction may cumulatively result in a clinically relevant attenuation of endothelial vasoprotection (Black and Garbutt, 2002; Krantz et al., 1996; Poitras and Pyke, 2013). Evidence suggests that cortisol, a hormone released as a result of

hypothalamic pituitary adrenal (HPA) axis activation, plays a mechanistic role in post-stress endothelial dysfunction (Akaza et al., 2010; Baykan et al., 2007; Broadley et al., 2005; Plotnick et al., 2017). Broadley et al. (2005) found that the negative impact of acute mental stress on FMD was abolished when cortisol production was inhibited with administration of metyrapone. More recently, Plotnick et al. (2017) observed an inverse relationship between post-stress FMD changes and cortisol reactivity (those with greatest reactivity experienced the greatest FMD decline post-stress). However, a decline in FMD post-stress has also been observed in the absence of an increase in cortisol (Szijgyarto et al., 2013) suggesting that other mechanisms are also involved. It is well established that depression influences HPA axis function (Pariante and Lightman, 2008; Varghese and Brown, 2001); however,

Abbreviations: FMD, Flow-mediated dilation; SR, Shear rate; TSST, Trier-social stress test; DSM-IV-TR, Diagnostic and statistical manual of mental disorders – fourth edition (text revision); HRSD, Hamilton rating scale for depression; SCID-I/P, Structured clinical interview for DSM-IV axis disorders ⁎ Corresponding author at: Cardiovascular Stress Response Laboratory, School of Kinesiology and Health Studies, Queen's University, Kingston K7L 3N6, ON, Canada. E-mail address: [email protected] (K.E. Pyke). https://doi.org/10.1016/j.ijpsycho.2018.12.003 Received 14 May 2018; Received in revised form 26 November 2018; Accepted 3 December 2018 Available online 06 December 2018 0167-8760/ © 2018 Elsevier B.V. All rights reserved.

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(i.e., type, dose) or psychotherapy (i.e., frequency of sessions) in the previous 3 months, and a pre-existing medical condition that could interfere with participation in physical activity (e.g., cardiovascular disease, untreated hypertension, etc.). Participants were not eligible if they were currently (or had regularly in the past 2 years) participated in exercise or yoga classes. Women who were pregnant or actively trying to become pregnant were also excluded. Forty-three women recruited from Queen's University and the larger surrounding community of Kingston, Ontario met the above criteria and were included in the study. Participants were instructed to abstain from food and cigarette smoking for 6 h, to avoid alcohol and caffeine for 12 h and exercise for 24 h prior to the experimental visit.

little research has investigated the impact of depression on vulnerability to acute stress-induced endothelial dysfunction. In a study of women in remission from major depressive disorder, Wagner et al. (2012) found that FMD was impaired following a mental stress task; however, cortisol responses were not assessed. No study to date has examined the impact of acute mental stress on endothelial function, and how this may relate to cortisol reactivity, in individuals with a current diagnosis of depression. The majority of the evidence suggests that cortisol reactivity to acute stress is blunted in individuals diagnosed with depression (Burke et al., 2005; Zorn et al., 2017). The degree of blunting appears to be related to depression severity, with greater severity being associated with more pronounced blunting (Burke et al., 2005; de Rooij et al., 2010). Given the evidence supporting a mechanistic role of cortisol in post-stress FMD impairment (Broadley et al., 2005), it is possible that the impact of acute stress on FMD in individuals with depression varies depending on the magnitude of cortisol reactivity such that blunted reactivity is associated with less pronounced post-stress FMD impairment. Therefore, the purpose of this investigation was two-fold. The primary objective was to examine the impact of acute mental stress on FMD in women with a current diagnosis of a depressive disorder, the majority of whom met DSM-IV-TR (American Psychiatric Association, 2000) criteria for major depressive disorder. The secondary objective was to identify whether stress-induced changes in FMD were related to cortisol reactivity. We hypothesized that FMD would be impaired following acute mental stress and that the degree of impairment would be greater in women with more pronounced cortisol reactivity to the stress task. Investigating the impact of acute mental stress on endothelial function and its relationship with cortisol reactivity in depression is important to further our understanding regarding the link between HPA axis function and cardiovascular risk in this population.

2.2. Experimental design Participants came to the laboratory for one experimental visit 1–7 days following their in-person psychological interview. The experimental visit was conducted between 3:00–5:00 pm to control for diurnal variations in cortisol (Weitzman et al., 1971). Upon arrival at the laboratory, participants were asked to lie supine for 35 min. At the end of the rest period, the first FMD trial was performed (pre-stress FMD; Fig. 1), which was followed by the administration of the Trier Social Stress Task (TSST). Fifteen minutes following the completion of the TSST, a second FMD trial was conducted (Post-Stress FMD; Fig. 1). 3. Experimental procedures 3.1. Clinical depression diagnostic criteria The Structured Clinical Interview for DSM-IV Axis I Disorders (SCIDI/P; First et al., 2002) was administered to assess current and past DSMIV-TR diagnoses. The SCID-I/P has demonstrated excellent reliability and validity (Williams et al., 1992). During the telephone screening interview, the Mood Module of the SCID-I/P was administered to determine initial eligibility. The full SCID-I/P was administered to assess psychiatric history prior to the laboratory visit.

2. Methods 2.1. Participants and screening protocol The study protocol was approved by the Health Sciences Research Ethics Board at Queen's University. The present study was a part of a larger trial examining the efficacy of yoga versus aerobic training in the treatment of depression. All interested participants were assessed for eligibility using a telephone screening interview, which was followed by an in-person psychological assessment conducted prior to the preintervention vascular testing. Inclusion criteria consisted of being female, between the ages of 18 and 65 years, and meeting DSM-IV-TR (American Psychiatric Association, 2000) criteria for a current episode of non-chronic, unipolar depressive disorder (i.e., major depressive disorder, adjustment disorder with depression mood, depressive disorder not otherwise specified). Exclusion criteria included the presence of a psychotic disorder, bipolar disorder, substance dependence, current suicidality beyond simple ideation, a medical condition that could be the cause of depression, and/or a change in antidepressant medication

3.2. Depressive symptoms assessment The Hamilton Rating Scale for Depression (HRSD; Hamilton, 1960) is a 17-item semi-structured interview that was used to assess the severity of depressive symptoms in the past week. Higher scores indicated more severe depressive symptoms, with cut-off scores for severity as follows: 0–7 = normal/absence of symptoms; 8–16 = mild depression; 17–23 = moderate depression; > 23 = severe depression (Zimmerman et al., 2013). The HRSD has largely been regarded as the “gold standard” measure of depression severity (Demyttenaere and De Fruyt, 2003; Williams, 2001), with adequate psychometric properties (Bagby et al., 2004).

Fig. 1. Protocol timeline. Brachial artery flow-mediated dilation (FMD) was assessed immediately before and 15 min after administering the Trier Social Stress Test (TSST- acute stress). Saliva samples were collected for cortisol determination at 8 separate time points. S1–S8, Sample 1–Sample 8. 114

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3.3. Anthropometric measures Measurements of height and weight were obtained for the calculation of body-mass index using the equation: BMI = weight (kg)/ height2(m). Age at the time of study participation was also recorded.

cortisol responses (Dickerson and Kemeny, 2004). Throughout both tasks, participants were told to continue talking, maintain eye contact with panel members, and remain still. Upon study completion, participants were debriefed regarding the purpose of the task and the nature of the deception.

3.4. Cardiorespiratory fitness assessment

3.9. Saliva sampling

Participants were asked to complete a widely-used submaximal exercise test (YMCA Submaximal Cycle Ergometer Test; described in Golding and Sinning, 1989), which allowed for an estimate of maximal oxygen uptake (VO2 peak) based on the heart rate versus cycle ergometer power output extrapolated to age predicted maximal heart rate (220-age). The exercise test occurred after vascular testing was completed.

Saliva samples were obtained by having participants saturate but not chew on a synthetic saliva collector (SARSTEDT Salivette) for approximately 2–3 min or until the swab was saturated. To adequately capture the time-course of cortisol reactivity to the stress task, salivary samples were obtained at time-points outlined by Kirschbaum et al. (1993) with the inclusion of two additional samples (S1 and S8; Fig. 1). After a 35-min rest period, participants provided two saliva samples 5min prior to- and immediately before the pre-stress FMD (S1 and S2, respectively; Fig. 1). No significant differences were observed between these two samples (Z = −1.532, p = 0.127); thus, the sample taken immediately prior to the pre-stress FMD (S2, Sample 2; Fig. 1) is referred to as the baseline sample. An additional 6 samples were collected throughout the experimental protocol, including a sample immediately following completion of the TSST (S4, Sample 4; Fig. 1) and a poststress FMD sample (S6, Sample 6; Fig. 1).

3.5. Subject monitoring Heart rate (HR) and blood pressure (BP) were measured continuously throughout the experimental visit. A 3‑lead electrocardiogram was used to obtain HR. Blood pressure was monitored using an automated blood pressure device (Finometer PRO, Finapres Medical Systems, Amsterdam, The Netherlands). These signals were recorded in LabChart (AD Instruments, Colorado Springs, CO) for future analysis.

4. Data analysis

3.6. Brachial artery blood velocity and diameter

4.1. Stress reactivity

Brachial artery blood velocity was collected using Doppler ultrasound operating at 4 MHz (Vivid i2 GE Medical Systems). The Doppler shift frequency spectrum was analyzed using a Multigon 500P TCD spectral analyzer (Multigon Industries, Yonkers, N.Y., USA) to determine the mean blood velocity, and the resulting voltage output was continuously recorded for subsequent analysis (LabChart, AD Instruments, Colorado Springs, CO). Brachial artery diameter was collected using 2D ultrasound (12 MHz in B mode, Vivid i2 Medical systems) with an insonation angle of 68° as previously described (Pyke et al., 2008). Ultrasound images were recorded using a VGA to USB frame grabber (Epiphan systems Inc.) and saved as .avi files on a separate computer (Camtasia Studio, TechSmith Okemos, MI, USA) as previously described (Jazuli and Pyke, 2011).

To characterize stress reactivity, HR, mean arterial pressure (MAP), and salivary cortisol were analyzed. HR and MAP were analyzed offline in LabChart (AD Instruments, Colorado Springs, CO) and compiled into 1-min average time bins. Baseline pre-stress HR and MAP were defined as the 1-min average collected during the baseline minute of the prestress FMD trial. Cardiovascular reactivity to the stress task was determined as the difference between the pre-stress baseline and the peak 1-min average of HR and MAP during the TSST (Fang et al., 2014; Yim et al., 2010). Timing of individual peak response varied, and this approach avoids underestimating reactivity by averaging individual responses at each time point. Blood pressure recordings were not collected for seven participants due to equipment failure; thus, MAP was reported for the remaining 36 participants. Reactivity change scores for HR and MAP (i.e., ∆HR, ∆MAP), defined as peak levels during the TSST minus baseline, were also calculated for correlation analyses. Salivary cortisol swabs were centrifuged to extract saliva to be frozen for storage at −80 °C. At the time of the analyses, samples were thawed to room temperature, spun at 380g for 15 min and assayed in duplicate using an enzyme-linked immunoassay (ELISA) kit according to the kit instructions (No. 1–3002; Salimetrics, State College, PA, USA). Cortisol reactivity was determined by comparing baseline cortisol (Sample 2, S2; Fig. 1) to the peak level that occurred during/following the TSST (i.e., highest of S3-S8; Fig. 1). The change in cortisol (∆CORT) was defined as the peak cortisol minus baseline and was calculated for correlation analysis. In two participants, cortisol data were missing and, as such, cortisol was reported in the remaining 41 participants.

3.7. Brachial artery flow mediated dilation (FMD) via reactive hyperemia A standard FMD test was performed twice on the left arm (Fig. 1). An occlusion cuff was placed at the antecubital fossa, which was distal to the location of the brachial artery ultrasound probe. Both brachial artery blood velocity and diameter were recorded for 1 min prior to cuff inflation to a pressure of 250 mm Hg for 5 min. Brachial artery blood velocity and diameter were then recorded for 1 min prior to and 3 min following cuff release. 3.8. Trier Social Stress Test (TSST) The TSST was administered by an unfamiliar panel of two laboratory personnel (Kirschbaum et al., 1993). Participants were given 10 min to prepare a 5-min speech explaining why they were a suitable candidate for a job position of their choosing. Participants were informed that their speech was being videotaped for analysis of nonverbal behaviour. Immediately following the speech task, participants underwent a mental arithmetic task in which they were asked to serially subtract 13, starting from 2083, as quickly as possible without making mistakes. Periodically, participants were told that their answer was incorrect (even if correct) and to start again from 2083. This increases the uncontrollability of the task which has been shown to elicit greater

4.2. Brachial artery blood velocity and diameter Brachial artery blood velocity was analyzed offline in 3-s average time bins using data acquisition software (LabChart, AD Instruments), as described by Pyke et al. (2008). Brachial artery diameter was analyzed using automated edge-detection software (Encorder FMD & Bloodflow v3.0.3, Reed Electronics), as previously described (Jazuli and Pyke, 2011). The diameter data was compiled in 3-s time bins, and this was used to calculate shear rate (described below).

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4.3. Shear rate

Table 1 Participant characteristics.

Shear rate (SR) was calculated as brachial artery blood velocity divided by diameter (Gnasso et al., 2001), with the time aligned velocity and diameter 3-s time bins. The SR stimulus was quantified as the SR area-under-the-curve to peak brachial artery diameter (SR-AUC). In nine participants, velocity signals were lost and, as such, the SR-AUC is reported for the remaining 34 participants. 4.4. Flow-mediated dilation A single, blinded investigator (KD) analyzed all images. Absolute change (absFMD) and percent change (%FMD) in diameter were computed for both pre- and post-stress FMD trials. Absolute FMD was calculated as the difference in arterial diameter (in mm) from baseline to the peak 3-s average diameter time bin post-cuff release, and %FMD was calculated as the percent difference between these same values. Additionally, change in %FMD from pre- to post-stress (∆%FMD; calculated as post- minus pre-stress %FMD) was calculated for correlation analyses. Due to poor wall tracking during baseline, the diameter in the last minute of cuff occlusion was substituted for baseline diameter for two participants in the pre-stress FMD trial and two participants in the post-stress FMD trial.

Variable

Total (N = 43)

Age (yrs) BMI (kg/m2) Estimated VO2 peak (ml·kg−1·min−1) Ethnicity Caucasian Asian Other Education Grade 12 Some college/university Undergraduate/college degree Graduate degree Smoking history Cigarettes per day HRSD Number of depressive episodes Medication use

33 ± 14 26.3 ± 5.2 29.5 ± 6.5 32 (74.4) 6 (14.0) 5 (11.6) 1 (2.3) 21 (48.8) 13 (30.2) 8 (18.6) 3 (7.0) 7.3 ± 3.2 13 ± 4 3.5 ± 2.6 16 (37.2)

Note. Age and BMI are mean ± SD. Ethnicity, education, smoking history and medication use are n (% of sample). BMI, body mass index; HRSD, Hamilton Rating Scale for Depression [0–7: normal/absence of symptoms; 8–16: mild depression; 17–23: moderate depression; > 23: severe depression].

4.5. Statistical analysis

experienced their peak cortisol levels before or during the post-stress FMD test.

Statistical analyses were conducted using SigmaPlot 11 software (Systat Software Inc., San Jose, CA) or IBM SPSS (SPSS Inc., Chicago, IL, USA). All values are expressed as the mean ± the standard deviation (SD). Significance was set at p < 0.05. To test the primary hypothesis, paired t-tests were used to compare pre-versus post-stress %FMD and absFMD. To assist in interpretation of the FMD response, bivariate correlation analysis was also used to assess the association between the shear stress stimulus and the FMD response as follows: (1) SR-AUC and FMD in each FMD trial; and (2) the change in SR-AUC and the change in FMD from pre- to post-stress. In addition, to account for a change in baseline diameter from pre to post-stress, a linear mixed model was used to compare FMD pre- and post-stress with baseline diameter entered as a covariate. Paired t-tests were used to assess the impact of acute mental stress on HR, MAP, cortisol, and SR-AUC. If the Shapiro-Wilk normality test failed, Wilcoxon Signed Rank test analysis was performed. To test the secondary hypothesis, bivariate correlation analysis was used to assess the association between cortisol reactivity (∆CORT) and Δ%FMD from pre- to post-stress. Bivariate correlation analyses were also used to determine associations between cardiovascular reactivity (∆HR, ∆MAP) and ∆%FMD, between ∆%FMD and HRSD scores, between ∆CORT and HRSD scores and between cardiorespiratory fitness and pre-stress % FMD, ∆%FMD, ∆CORT, ∆MAP, and ∆HR. Cook's Distance test was used to identify outliers (Henderson, 2006).

5.3. Baseline HR and MAP during the FMD tests Hemodynamic variables measured during baseline of the two FMD tests are shown in Table 2. Baseline HR was not significantly different pre-versus post-stress (Z = −0.0604, p = 0.957). Baseline MAP was significantly higher in the post-stress FMD trial (Z = 4.292, p < 0.001). 5.4. Shear rate Neither baseline SR nor SR-AUC were significantly different from pre- to post-stress (Table 2 and Fig. 3a, respectively). 5.5. Baseline diameter and FMD Both baseline and peak brachial artery diameter significantly decreased from pre- to post-stress (Z = −5.035, p < 0.001 and t (42) = 6.804, p < 0.001, respectively; Table 2). However, no changes in %FMD or absFMD were observed (Fig. 3b and Table 2, respectively). Similarly, no significant differences in %FMD or absFMD from pre- to post-stress were observed when baseline diameter was added as a covariate (F(1, 83) = 0.015, p = 0.904 and F(1,83) = 0.015, p = 0.903, respectively). Given the similarity in %FMD and absFMD analyses, only %FMD is reported in the correlational analyses below. Time to peak diameter also did not differ between trials (Table 2). Within each FMD trial, SR-AUC was modestly related to the %FMD response (pre-stress: R = 0.696, p < 0.001; post-stress: R = 0.551, p < 0.001); however, the addition of the SR-AUC as a covariate did not reveal a change in FMD from pre- to post-stress (F(1,71) = 1.388, p = 0.243).

5. Results 5.1. Participant characteristics Baseline participant characteristics are reported in Table 1. 5.2. Responses to the stress task Heart rate, blood pressure and cortisol all increased significantly from baseline to peak in response to the TSST (Fig. 2). The timing of peak stress induced responses varied between participants. For cortisol reactivity the largest percentage of participants demonstrating the greatest increase in cortisol in sample 3 (37%), followed by sample 4 (22%) and sample 8 (20%). Overall, 78% of the participants

5.6. Stress reactivity correlation analysis A moderate negative correlation was observed between ∆%FMD and ∆CORT from pre- to post-stress (F(1, 39) = 7.102, R = −0.392, p = 0.011; Fig. 4). Examination of the correlation between ∆%FMD and ∆CORT revealed two outliers. When these participants were removed, 116

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Fig. 2. Heart rate (HR) (A), mean arterial pressure (MAP) (C), and cortisol (E) at baseline and peak post Trier Social Stress Test (TSST) onset (peak values are the average of the highest post TSST onset value as individually determined for each participant). The time course of HR (B) and MAP (D) and cortisol (F) responses are represented for descriptive purposes. BSL; baseline.

the relationship trended towards significance (F(1, 37) = 3.875, R = 0.308; p = 0.057). No significant correlations were observed between ∆%FMD and ∆HR (F(1, 39) = 1.780, R = 0.209, p = 0.190) or ∆ %FMD and ∆MAP (F(1,34) = 0.719, R = 0.144, p = 0.403). Furthermore, neither ∆%FMD nor ∆CORT correlated with the HRSD scores (F (1, 41) = 0.00133, R = 0.00570, p = 0.971 and (F(1, 39) = 0.846, R = 0.146, p = 0.363, respectively). In addition, age did not relate to ∆CORT or change in %FMD with stress (F(1, 39) = 0.0863, R = 0.470, p = 0.771 and F(1,41) = 0.00000837, R = 0.000452, p = 0.998, respectively). Finally, cardiorespiratory fitness did not correlate with prestress %FMD (F(1,39) = 0.056, R = 0.038, p = 0.814), ∆%FMD (F (1,39) = 0.313, R = 0.089, p = 0.579), ∆CORT (F(1,37) = 0.163, R = 0.066, p = 0.689), ∆MAP (F(1,32) = 0.061, R = 0.043, p = 0.807), or ∆HR (F(1,37) = 0.001, R = 0.006, p = 0.970).

Table 2 Hemodynamic, diameter and shear rate variables during the flow-mediated dilation (FMD) tests. Variable

Pre-stress

Post-stress

t

Baseline MAP (mm Hg) Baseline heart rate (beats·min−1) Baseline shear rate (s−1) Baseline diameter (cm) Peak diameter (cm) Time-to-peak diameter (s) Absolute FMD (cm)

96.6 ± 12.3 65.1 ± 11.0

103.2 ± 14.4 4.292 < 0.001 64.6 ± 10.0 −0.0604 0.957

21.2 ± 12.6 0.289 ± 0.04 0.311 ± 0.04 47.0 ± 17.0 0.022 ± 0.01

19.3 ± 7.87 0.280 ± 0.04 0.302 ± 0.04 49.6 ± 23.9 0.022 ± 0.01

−0.162 −5.035 6.804 0.138 0.0575

p

0.878 < 0.001 < 0.001 0.896 0.954

Note. Values are mean ± SD. MAP, mean arterial pressure; FMD, flow-mediated dilation. Italics indicate Z-statistic reported (Wilcoxon signed rank test was performed).

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6.1. Impact of acute mental stress on FMD Research investigating the relationship between acute mental stress and endothelial function in individuals with depression is limited. In a study of postmenopausal women in full remission from a lifetime history of depression, Wagner et al. (2012) reported a decrease in brachial artery FMD 20 min post-stress. Findings from the present study, demonstrating no significant differences in FMD from pre- to post-stress, are in contrast to this previous investigation and our predictions. To understand these discrepant findings, differences in participant characteristics in the present study versus Wagner et al. (2012) are important to consider. For instance, women included in the Wagner et al. (2012) study were in full remission from depression, with an average of 11 years since the last major depressive episode. In contrast, the present study required participants to meet DSM-IV-TR criteria for a current depressive disorder. This observation, along with the considerable difference in age between the samples (mean = 33 vs. 60 yrs), suggests that both depression history and age may influence endothelial susceptibility to acute mental stress. In the present study, however, neither age nor HRSD score influenced the magnitude of FMD change. Differences in the acute stress tasks used in this study and in Wagner et al.'s (2012) study are also worth noting. Participants in the Wagner et al. (2012) study underwent a 5-min serial subtraction mental arithmetic task. Our protocol involved a similar mental arithmetic task in addition to having participants prepare and deliver a 5-min speech task. The social evaluative threat in our study was also enhanced with the addition of video recording (Dickerson and Kemeny, 2004). Social evaluation threat is known to enhance cortisol responses in acute stress tasks (Dickerson and Kemeny, 2004). It is possible that differences in stress tasks resulted in meaningful differences in cortisol reactivity; however, this cannot be verified due to the absence of cortisol measurements in the Wagner et al. (2012) study. Even if the stress task utilized in Wagner et al. (2012) did not maximize activation of the HPA axis, the response to their stress task was sufficient to impair FMD.

Fig. 3. (A) Shear rate area under the curve (AUC) until peak diameter post-cuff release. (B) %FMD pre- and post-Trier Social Stress Test (TSST).

6.2. Cortisol responses to acute stress and the association between cortisol reactivity and FMD The baseline cortisol levels observed in the current study are comparable to those found in healthy individuals without depression (~6 nmol/L; Vining et al., 1983; Ghiadoni et al., 2000; Zimmer et al., 2003; Ljubijankić et al., 2008; Szijgyarto et al., 2013; Stephens et al., 2016; Plotnick et al., 2017). Our findings run counter to those of previous investigations that have generally demonstrated elevated basal cortisol levels in adults with depression (McKay and Zakzanis, 2010; Pariante and Lightman, 2008). These discrepant findings may be owing to the mild severity level of depression symptoms in our communityderived sample. In agreement with the small cortisol reactivity observed in the present study, evidence suggests that women with depression demonstrate blunted cortisol reactivity to psychological challenges when compared to controls (Burke et al., 2005; Zorn et al., 2017). Indeed, although a significant cortisol response to the TSST was observed in the present study (individually determined peak value post-TSST onset greater than baseline Fig. 2E), the overall trajectory was quite flat (Fig. 2F) and the mean magnitude of the peak change was lower than previous investigations of the impact of the TSST on cortisol reactivity in healthy individuals, including samples of healthy women who may have lower reactivity compared to men (Ghiadoni et al., 2000; Kirschbaum et al., 1993; Plotnick et al., 2017; Stephens et al., 2016; Szijgyarto et al., 2014). For example, in healthy young (18–30 yrs) women, Stephens et al. (2016) observed a mean cortisol reactivity to the TSST almost double that observed in the present study (~∆2.1 nmol/L vs. ∆1.2 nmol/L, respectively). It is possible that the small cortisol reactivity in this sample of

Fig. 4. Bivariate correlation analysis between change in cortisol and change in %flow-mediated dilation (%FMD) from baseline to peak-reactivity following the Trier Social Stress Test (TSST). Cook's distance analysis identified two outliers (point circled by dotted lines). With the removal of these points the strength of the relationship decreased (R = 0.308; p = 0.057).

6. Discussion This study provides the first investigation of FMD responses to acute mental stress in women with clinical depression. The primary finding was that, contrary to our hypothesis, acute mental stress did not result in FMD impairment. However, we observed a modest negative correlation between cortisol reactivity and change in FMD from pre- to poststress, such that larger increases in cortisol were associated with greater decreases in the post-stress FMD response. These findings suggest that although acute mental stress did not result in significant impairment in endothelial function in our sample of women with depression, greater cortisol reactivity may augment individual vulnerability to post-stress endothelial dysfunction. 118

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women contributed to the absence of an effect of acute mental stress on FMD. Although, as described in the introduction, a small post-stress decline in FMD has been found in the absence of an increase in cortisol (Szijgyarto et al., 2013). There is ample evidence supporting mechanistic involvement of cortisol in post-stress FMD impairment. The most prominent evidence is provided by Broadley et al. (2005a) who observed that inhibition of cortisol production with metyrapone prevented post-stress FMD impairment. The mechanistic role of cortisol in acute stress induced endothelial dysfunction may be mediated by cortisol induced reductions in nitric oxide (NO) bioavailability via the direct inhibition of the enzyme responsible for endothelium derived NO synthesis (eNOS) and/or indirectly by enhancing the production NOscavenging reactive oxygen species (reviewed in Poitras and Pyke, 2013). Furthermore, Broadley et al. (2006) found that compared to placebo, metyrapone administration improved endothelial function in adults with depression, suggesting that cortisol is an important determinent of FMD in this population. Taken together, these findings suggest that high cortisol reactivity and thus, hightened cortisol exposure, may increase vulnerability to FMD impairment following acute stress. Our observation of a moderate negative association between ∆% FMD and ∆CORT (Fig. 4) suggests that cortisol reactivity may play some role in the effects of mental stress on FMD. Plotnick et al. (2017) observed a similar but stronger (R = 0.809) relationship between cortisol reactivity and TSST-induced changes in FMD in healthy male participants; although, this finding is not unanimous (Ghiadoni et al., 2000). We observed substantial between participant variability in cortisol reactivity and there is evidence suggesting that cortisol responses to stress vary with depression severity (Burke et al., 2005a; Weinstein et al., 2010). However, in the present study neither cortisol reactivity nor post-stress changes in FMD correlated with HSRD score, suggesting that factors other than depression severity underlie the observed variability in these responses. As always when correlating change scores, it is possible that exaggerated measurment error interfered with the detection of associations. However, the modest strength of the relationship between percent change in FMD from pre- to post-stress and cortisol reactivity in our study indicates that there may be additional important factors involved in determining any influence of acute stress on FMD in women with depression. Antidepressant medications can influence cortisol reactivity (Vermetten et al., 2006); however, in the present study neither cortisol reactivity nor FMD responses differed between participants who were and were not taking medication (data not shown).

responses to stress. However, %FMD magnitude in the present study (pre-stress FMD: 7.8 ± 3.8%) is similar to the %FMD in a group of healthy young women recently reported by our group (7.9 ± 4.3% and 6.4 ± 3.1% in the early and late follicular phases, respectively; D'Urzo et al., 2017). This suggests an absence of endothelial dysfunction at baseline. Although the present study relied on within-subjects comparisons, neither menstrual nor menopausal status at the time of testing were determined, which may influence vulnerability to acute impairment (Luca et al., 2016) and baseline endothelial function (Adkisson et al., 2010; Moreau et al., 2012), respectively. Therefore, it is possible that the observed variability in FMD changes following acute stress relates to the effects of menstrual phase and/or menopause status. Finally, as mentioned above, this investigation reported on only a portion of a larger intervention study that required multiple in-person assessments and regular participation in yoga or aerobic exercise classes. Given the large time commitment already required by the larger study, a time control was not feasible. If a change in FMD occurred over time due to diurnal variation or other factors, that may have masked a change in FMD in response to stress. However, our group and others have previously demonstrated the stability of FMD across multiple trials in healthy (Harris et al., 2006; Pyke and Jazuli, 2011) and depressed men and women (Broadley et al., 2006). Therefore, it is unlikely that the absence of a time control condition impacted the interpretation our findings.

6.3. Limitations

The authors would like to thank all the participants for their time, patience and enthusiasm throughout this study. This research was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to K.E. Pyke and a Senate Advisory Research Council pilot grant to K.L. Harkness. T. J. King, M.D. Plotnick and J. S. Williams were funded by NSERC post graduate scholarships. C. L. La Rocque was funded by a Canadian Institute of Health Research post graduate scholarship.

7. Summary and conclusion This study is the first investigation of the impact of acute mental stress on FMD in women experiencing a current episode of depression. We did not detect a negative impact of stress on FMD, which is in contrast to some previous reports of an attenuated FMD response following acute mental stress in healthy individuals (Broadley et al., 2005; Ghiadoni et al., 2000; Spieker et al., 2002) and those with a history of major depression (Wagner et al., 2012). Taken together, these findings indicate that acute mental stress does not uniformly decrease endothelial function in women diagnosed with clinical depression; however, the relationship between cortisol reactivity and changes in FMD suggests that larger cortisol responses to stress may increase vulnerability to post-stress endothelial dysfunction in this population. Further research is required to fully elucidate the interaction between acute stress, cortisol, and endothelial function in adults with depression. Acknowledgements

In this study, FMD was measured immediately prior to- and 15-min following acute mental stress. This methodology was informed by previous reports of a significant FMD decline from immediately to 90 min following acute mental stress (Broadley et al., 2005; Ghiadoni et al., 2000; Jambrik et al., 2004; Spieker et al., 2002). However, the time course of endothelial function decline has not been investigated in a population with depression. Therefore, our protocol may have failed to capture an overall impairment that occurred earlier or later than the single post-stress FMD assessment. Furthermore, it is possible that differences in the time course of FMD decline may relate to the time course of cortisol reactivity. Indeed, only 10% of participants experienced their peak increase in cortisol during the post-stress FMD test, and 22% did not exhibit their peak increase in cortisol until after the post-stress FMD test (samples 7 or 8). Future investigations would benefit from the inclusion of additional post-stress FMD assessments to allow for better alignment between individual peak cortisol concentrations and the FMD response. Additional limitations include the absence of a non-depressed control group and time control condition. We cannot evaluate potential depressed versus non-depressed differences in FMD and cortisol

Author contributions C.L.L., K.L.H., and K.E.P. conceived and designed the research question and protocol. C.L.L., J.S.W., T.J.K. and T.J.R.S. performed data collection; K.A.D., J.S.W. and K.E.P analyzed cardiovascular data. T.J.K and M.D.P. analyzed the cortisol data under the supervision of B.J.G. K.A.D and J.S.W. prepared figures; K.A.D drafted the manuscript; and all authors edited and revised the manuscript and approved the final version of the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. 119

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