Peripheral blood mononuclear cell mitochondrial bioenergetics are related to vascular endothelial function in young and older adults

Abstract

The use of peripheral blood mononuclear cells (PBMCs) in cardiovascular research is increasingly common. However, little is known regarding potential age-related changes in mitochondrial bioenergetics and oxidative stress in PBMCs, or whether such changes relate to endothelial function. We assessed mitochondrial bioenergetics and antioxidant buffering capacity (AoxBC) capacity in PBMCs from young (n=18; 21±2 yrs) and older (n=17, 66±4 yrs) adults. High-resolution respirometry and fluorometry measured mitochondrial respiration rate (JO₂) and membrane potential (Δψm), respectively, in response to substrate provision (pyruvate/glutamate/malate/succinate; PGMS) and a bioenergetic creatine kinase (CK) clamp at physiological ATP:ADP ratios (PCr1, PCr2, and PCr3). MtROS emission was measured as hydrogen peroxide (H₂O₂) emission, and H₂O₂ production was quantified using inhibitors of glutathione reductase and thioredoxin/peroxiredoxin. AoxBC was calculated as the percentage of H₂O₂ produced but not emitted. Endothelial function was assessed via flow-mediated dilation (FMD). JO₂ was similar between groups at baseline (p=0.08) and lower energetic states (PCr2, PCr3; p≥0.09), but was lower in older adults at higher energetic states (PCr1: 14.05±2.11 vs. 12.03±2.98 pmol·sec⁻¹·10⁶ cells⁻¹, p=0.03; PGMS: 20.61±2.11 vs. 16.58±3.56 pmol·sec⁻¹·10⁶ cells⁻¹; p=0.0009). Δψm was hypo-polarized in older compared with young adults at all energetic states (p≤0.003). Although there were no statistical differences in H₂O₂ emission (p=0.43) or production (p=0.18), AoxBC was lower in older adults (52.59±15.44% vs. 63.49±10.30%; p=0.03). Age-related changes in JO₂ (PGMS, p=0.02) and Δψm (PGMS, p=0.0008; PCr2, p=0.04; PCr3, p=0.02) were related to FMD. These data demonstrate associations between altered PBMC mitochondrial bioenergetics and age-related vascular endothelial dysfunction.

NEW AND NOTEWORTHY

The use of peripheral blood mononuclear cells (PBMCs) is increasingly common in cardiovascular research. However, relatively little is known regarding potential age-related changes in PBMC mitochondrial bioenergetics and oxidative stress, or whether age-related changes are related to endothelial (dys)function. We demonstrate altered mitochondrial bioenergetics (oxygen consumption rates and membrane potential) and antioxidant buffering capacity in PBMCs from older compared with young adults. We additionally demonstrate associations between mitochondrial bioenergetics and endothelial function (brachial-artery flow-mediated dilation).

Graphical Abstract

INTRODUCTION

Immune system dysregulation may be an important contributor to the development of hypertension and overt cardiovascular disease) by promoting a pro-inflammatory state and increased oxidative stress (1–3). As a constituent of whole blood, peripheral blood mononuclear cells (PBMCs) continually interact with the vascular endothelium; therefore, increased inflammation and oxidative stress from PBMCs may act on the endothelium and induce endothelial dysfunction. Accordingly, the use of PBMCs in clinical cardiovascular research (4–6) and in the investigation of mechanisms underlying vascular endothelial dysfunction has become increasingly common in recent years (7–10).

Age-related changes in mitochondrial bioenergetics and oxidative stress (11, 12) may be implicated in the development of vascular dysfunction, hypertension, and cardiovascular disease (13). In aged adults, alterations in mitochondrial bioenergetics and membrane potential contribute to increased mitochondria-derived reactive oxygen species (mtROS) production (14). An imbalance between mtROS production and antioxidant buffering capacity (AoxBC) may have deleterious impacts on vascular endothelial function (15, 16) by reducing NO bioavailability through the uncoupling of the endothelial NO synthase (eNOS) dimer and/or by directly depleting bioavailable NO through its interaction with superoxide radicals, resulting in the production of peroxynitrite (another potent source of oxidative stress) (17).

Previous work has demonstrated poorer PBMC mitochondrial respiration (JO₂) in early-stage heart failure (18) and frail older adults (19). Additionally, cardiometabolic risk factors [i.e., low-density lipoprotein (LDL)-cholesterol and systolic blood pressure (SBP)] were negatively associated with PBMC mitochondrial bioenergetics in mostly-healthy individuals across the adult lifespan, such that higher LDL-cholesterol and SBP were associated with poorer mitochondrial respiration (20). Another study demonstrated little-to-no age-related change in PBMC mitochondrial respiration (21). Other studies have demonstrated increased superoxide production in PBMCs of young adults for whom endothelial dysfunction is consistently observed (e.g., e-cigarette users and non-Hispanic Black men) (7, 9), and increased mtROS in T-cells of adults with major depressive disorder (10).

Relatively little work has been done to evaluate the impact of typical aging (i.e., generally-healthy but representative of the general population in terms of blood pressure, blood biochemistry, medication use, etc.) on mitochondrial bioenergetics and oxidative stress in PBMCs. Furthermore, there has yet to be an examination of real-time mtROS production and emission and AoxBC in PBMCs in young and/or older adults. Evaluating these real-time parameters of mtROS can provide specific insight into imbalances between mtROS production and clearance that may contribute to increased oxidative stress. Finally, it is unclear whether there is an association between potential age-related changes in PBMC mitochondrial function and vascular endothelial function.

The present study evaluated age-related differences in PBMC mitochondrial bioenergetics (JO₂ and membrane potential, Δψm) and oxidative stress. We employed a novel approach for the real-time assessment of mtROS production and emission and AoxBC in permeabilized PBMCs collected from young and older adults. We hypothesized that JO₂ and Δψm would be attenuated in older compared with young adults. Similarly, we hypothesized that mtROS production and emission would be greater, and that AoxBC would be poorer, in older compared with young adults. We also sought to evaluate whether mitochondrial bioenergetics, ROS production/emission, and/or AoxBC are correlated with brachial artery flow-mediated dilation (FMD), a commonly-used clinical assessment of vascular endothelial function that is prognostic for cardiovascular risk (22).

METHODS

Participants.

All experimental protocols were approved by the Institutional Review Board at the University of Georgia. Written and verbal consents were obtained from all subjects prior to participation, according to the Declaration of Helsinki. All participants underwent an initial screening that included a health history questionnaire, measures of height, weight, and BP. Participants were mostly-healthy young (18 – 26 yrs) and older (59+ yrs) adults. Subjects were non-diabetic (HbA1C ≤6.4%) with BMI <35 kg∙m². Normotensive and stage 1 hypertensive participants (SBP < 140 and DBP < 89 mmHg), and those with well-controlled hypertension, were included. All women in the older group were postmenopausal. Premenopausal women had naturally cycling (n = 6), were taking oral contraceptives (n = 4), or using alternative birth control methods (i.e., intrauterine device, NuvaRing; n = 2), and a urine pregnancy test confirmed the absence of pregnancy before experimental visits. To increase generalizability of our findings, women were tested without regard to menstrual cycle or oral contraceptive phase.

PBMC isolation.

Primary PBMCs were freshly isolated from 40 mL of venous blood collected in EDTA vacutainers. Whole blood samples were diluted and centrifuged using density gradient centrifugation at 650 g for 30 min at 10°C with slow acceleration and deceleration. The PBMC interface was removed, washed three times, and resuspended in sterile Dulbecco’s Phosphate-Buffered Saline.

PBMC mitochondrial respiration and membrane potential.

Immediately after PBMC isolation, JO₂ was measured using high-resolution respirometry (Oroboros Instruments, Innsbruck, Austria) as previously described (23, 24). These tests were performed with a volume of 0.5 mL at a concentration of 2.4 million cells per mL, resulting in an absolute volume of 1.2 million cells. A bioenergetic creatine kinase (CK) clamp technique, in which cellular energy demand (Gibbs-free energy; ΔGATP) is manipulated using the enzymatic reaction of CK and phosphocreatine (PCr), was used to measure mitochondrial oxygen consumption rates and electron conductance through the electron transport system under a physiologically relevant energetic demand state (25–27). The CK clamp allows for testing of JO₂ and Δψm at different physiological ATP:ADP ratios (energetic demand states). After recording baseline JO₂, PBMCs were given substrate (5 mM pyruvate, 2 mM malate, 10 mM succinate, and 10 mM glutamate) with 20 U/mL CK, 1 mM PCr, and 5 mM ATP to elicit a maximal JO₂, after which cytochrome C (10 μM) was added to check mitochondrial integrity. Next, sequential PCr titrations (6, 11, 15 mM) were used to reduce ΔG_ATP to approximate resting conditions. Herein, we refer to each stage of the PCr titration in descending order of ΔG_ATP as PCr1, PCr2, and PCr3 (i.e., PCr1 represents the highest energy state of the PCr titrations). In addition to recording absolute JO₂, the relation between JO₂ and ΔG_ATP during the CK clamp and maximal respiration was evaluated as an index of respiratory conductance, reflecting the sensitivity of mitochondria to changes in energetic demand (28). JO₂ was normalized and presented as pmol O₂·min⁻¹·10⁶ cells⁻¹.

Δψm was measured fluorometrically in buffer Z containing 5 mM creatine, using a spectrofluorometer (FluoroMax Plus-C; Horiba Instruments Inc., Irvine, CA). The Δψm was assessed with tetramethylrhodamine methyl ester (TMRM) at 30°C, simultaneously with the CK clamp assay protocol and with constant stirring. TMRM excitation/emission [(572/590 nm)/(551/590 nm)] fluorescence is quenched, indicating an increased ratio of 572/551 with greater Δψm polarization. As such, the 572/551 ratio is reported here as arbitrary units (a.u.) given that, to our knowledge, there has been no report validating the conversion of the 572/551 ratio to millivolts in PBMCs.

To further assess the mitochondrial bioenergetic relationships, JO₂ values were plotted against their corresponding Δψm at each ΔG_ATP. This relationship is crucial as, in vivo, the energy generated from the proton motive force driven by Δψm determines the extent to which ATP synthase can displace the ATP/ADP ratio away from equilibrium. Thus, the ability of mitochondria to control the relationship between JO₂ and Δψm serves as a crucial indicator of respiratory efficiency and mitochondrial readiness in response to energy demand (referred to as bioenergetic efficiency in this paper).

PBMC mtROS emission and production and antioxidant buffering capacity.

We measured mtROS emission and production and AoxBC in a subgroup of 16 young and 12 older adults. These tests were performed with a volume of 0.6 mL at a concentration of 2.0 million cells per mL, resulting in an absolute volume of 1.2 million cells. The mtROS emission and production were measured in buffer Z supplemented with Amplex Ultrared (5 μM), Cu-Zn superoxide dismutase (25 units/mL), and horseradish peroxidase (1 U/mL) detection system of H₂O₂ (Ex:Em 565:600), as previously described (23). After a baseline measurement period, real-time mtROS emission was assessed by adding pyruvate, malate, succinate, and glutamate. Next, inhibitors of the glutathione reductase [100 μM carmustine; BCNU] and thioredoxin/peroxiredoxin (1 μM auranofin; AF) – enzymes that are involved in the reduction of H₂O₂ to H₂O – were added to determine the overall H₂O₂ production of the tissue (29, 30). The detectable H₂O₂ increases with AF/BCNU, highlighting the production of H₂O₂ that was being buffered by the AOX systems. Thus, AoxBC was calculated as the percentage of mtROS produced but not emitted [i.e., (H₂O₂ Produced – H₂O₂ Emitted) / H₂O₂ * 100]. H₂O₂ emission and production rates were normalized and presented as pmol H₂O₂·sec⁻¹·10⁶ cells⁻¹. This approach for quantifying AoxBC was previously validated in skeletal muscle (23, 29) and our group recently translated the use of the technique to PBMCs.

Flow-mediated dilation.

Brachial artery FMD was measured according to established guidelines (31, 32). All FMD measurements were performed by a single sonographer (STW) and took place in a quiet, temperature-controlled room. The study began after a ≥10-minute supine rest period. Continuous ultrasound and Doppler imaging was performed on the brachial artery with a 60° insonation angle, placed proximal to an inflatable cuff on the participant’s forearm. Brachial artery diameter was continuously recorded during 1 minute of baseline, 5 minutes of forearm cuff occlusion (220 mm Hg), and 3 minutes after cuff release. FMD was calculated as [(diameter_peak − diameter_baseline)/diameter_baseline × 100].

Statistical analyses.

All statistical analyses were performed using GraphPad Prism software (v. 10.5, GraphPad Software, San Diego, CA). Age differences in participant characteristics (e.g., BMI, blood pressure, cholesterol, etc.) were assessed using unpaired t tests.

Mitochondrial bioenergetic data (JO₂ and Δψm) were analyzed using 2-way mixed effects models to asses age (young vs. older) and phase (baseline, max, PCr1, PCr2, and PCr3) effects. An unpaired t test was used to evaluate potential age differences in respiratory conductance (the relation between JO₂ and ΔG_ATP). Additional unpaired t tests were used to assess age differences in mtROS emission and production and AoxBC, and FMD. Simple linear regression analyses were used to evaluate the relations between indices of PBMC mitochondrial function and FMD.

All data are presented as mean ± SD except for box-whisker plots, which depict median, minimum, maximum, and first and third quartile values.

RESULTS

Subject characteristics.

Subject characteristics and medication use are summarized in Table 1. In addition to age (by design), older adults had higher LDL cholesterol (p = 0.002) and HbA1c (all p ≤ 0.001) compared with young adults. Otherwise, there were no differences between groups.

Table 1.

Subject characteristics

Characteristic	Older	Young
n	17 (8 M, 9 F)	18 (6 M, 12 F)
Age (yr)	66 ± 4*	21 ± 2
BMI (kg∙m−2)	25 ± 4	23 ± 3
SBP (mmHg)	118 ± 8	114 ± 6
DBP (mmHg)	70 ± 7	72 ± 6
Total Cholesterol	199 ± 38	170 ± 21
HDL-C	57 ± 12	62 ± 10
LDL-C	119 ± 30*	88 ± 23
HbA1c	5.3 ± 0.3*	4.9 ± 0.2
Medications
ACE Inhibitor	2
Beta Blocker	2
SSRI	2	4
Statin	4
Estrogen Replacement	2
Hormonal Contraceptive		6

BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; ACE; angiotensin-converting enzyme; SSRI, selective serotonin reuptake inhibitor.

^*p < 0.05 compared with Young.

PBMC mitochondrial bioenergetics.

Baseline JO₂ was similar between older and young adults (Older, 13.93 ± 3.72 pmol·sec⁻¹·10⁶ cells⁻¹; Young, 14.48 ± 2.18 pmol·sec⁻¹·10⁶ cells⁻¹; p = 0.61; g = 0.18). JO₂ was lower in older compared with young adults at maximum (Older, 16.64 ± 3.67 pmol·sec⁻¹·10⁶ cells⁻¹; Young, 20.61 ± 2.11 pmol·sec⁻¹·10⁶ cells⁻¹; p = 0.0008; g = 1.34; Figure 1A) and at the highest energetic state during the CK clamp (PCr1: Older, 12.03 ± 2.98 pmol·sec⁻¹·10⁶ cells⁻¹; Young, 14.05 ± 2.11 pmol·sec⁻¹·10⁶ cells⁻¹; p = 0.03; g = 0.79; Figure 1B), but not during the two lower energetic states of the CK clamp (PCr2: Older, 10.36 ± 2.55 pmol·sec⁻¹·10⁶ cells⁻¹; Young, 11.68 ± 1.57 pmol·sec⁻¹·10⁶ cells⁻¹; p = 0.09; g = 0.63; PCr3: Older, 9.36 ± 2.51 pmol·sec⁻¹·10⁶ cells⁻¹; Young, 10.46 ± 1.62 pmol·sec⁻¹·10⁶ cells⁻¹; p = 0.17; g = 0.52). Respiratory conductance, an indicator of the mitochondrial capacity to ramp up or down JO₂ to match energetic demand, was lower in older compared with young adults (Older, 3.61 ± 1.06 a.u.; Young, 4.84 ± 1.46 a.u.; p = 0.005; g = 0.96; Figure 1C).

Figure 1.

Oxygen consumption rates (JO₂) at maximum (Panel A) and during the creatine-kinase clamp (Panel B), and respiratory conductance (i.e., the slope of the relation between JO₂ and ΔG_ATP; Panel C). Boxes in panels A and C represent first and third quartiles with median values denoted by the horizontal line, and whiskers indicate minimum and maximum observations. * P < 0.05 older compared with young; † P < 0.05 compared with PCr3 in both groups; ‡ P < 0.05 compared with PCr2 in both groups.

Baseline Δψm was lower in older compared with young adults (Older, 0.35 ± 0.02 a.u.; Young, 0.37 ± 0.02 a.u.; p = 0.001; g = 1.28). Likewise, Δψm was lower in older compared with young adults during maximal respiration (Older, 0.37 ± 0.02 a.u.; Young, 0.40 ± 0.02 a.u.; p = 0.0001; g = 1.61; Figure 2A) and throughout the CK clamp (PCr1: Older, 0.40 ± 0.02 a.u.; Young, 0.41 ± 0.01 a.u.; p = 0.003; g = 1.07; PCr2: Older, 0.40 ± 0.02 a.u.; Young, 0.42 ± 0.01 a.u.; p = 0.0007; g = 1.19; PCr3: Older, 0.40 ± 0.02 a.u.; Young, 0.42 ± 0.02 a.u.; p = 0.0005; g = 1.23; Figure 2B). Finally, there was a leftward and downward shift in the bioenergetic efficiency for older compared with young adults (Figure 2C).

Figure 2.

Mitochondrial membrane potential during maximum respiration (Panel A) and the creatine-kinase clamp (Panel B), and the relation between mtMP and JO₂ (an indicator of respiratory efficiency and mitochondrial readiness in response to energy demand; Panel C). Boxes in panel A represent first and third quartiles with median values denoted by the horizontal line, and whiskers indicate minimum and maximum observations. * P < 0.05 compared with young; # P < 0.05 compared with PCr2 in young adults only.

PBMC mitochondrial oxidative stress.

Mitochondrial H₂O₂ emission (Older, 0.67 ± 0.21 pmol·sec⁻¹·10⁶ cells⁻¹; Young, 0.67 ± 0.23 pmol·sec⁻¹·10⁶ cells⁻¹; p = 0.43; g = 0.02) and production (Older, 1.58 ± 0.47 pmol·sec⁻¹·10⁶ cells⁻¹; Young, 1.85 ± 0.49 pmol·sec⁻¹·10⁶ cells⁻¹; p = 0.18; g = 0.57) were not significantly different between older and young adults (Figure 3A). However, mitochondrial AoxBC was significantly lower in older (52.59 ± 15.44%) compared with young adults (63.49 ± 10.30%; p = 0.03; g = 0.84; Figure 3B).

Figure 3.

Mitochondrial hydrogen peroxide (H₂O₂) emission and production (Panel A) and antioxidant buffering capacity (Panel B) in PBMCs from young and older adults. Boxes represent first and third quartiles with median values denoted by the horizontal line, and whiskers indicate minimum and maximum observations. * P < 0.05 compared with young.

Associations between PBMC mitochondrial function and FMD.

As expected, FMD was significantly lower in older compared with young adults (7.72 ± 3.39% vs. 3.19 ± 2.20%, p = 0.0001; Figure 4). There was a significant relation between maximal OCR and FMD (p = 0.02; R² = 0.17; Figure 5A). Conversely, there was no relation between OCR and FMD at baseline (p = 0.18; R² = 0.06). Likewise, there were no relations between OCR and FMD at PCr1 (p = 0.14; R² = 0.07), PCr2 (p = 0.19; R² = 0.06), or PCr3 (p = 0.18; R² = 0.06) (Figure S1). When examined by age group, there were no correlations between OCR and FMD within either age group (all p ≥ 0.16).

Figure 4.

Age differences in flow-mediated dilation (FMD). Boxes represent first and third quartiles with median values denoted by the horizontal line, and whiskers indicate minimum and maximum observations. * P < 0.05 compared with young.

Figure 5.

Correlations between maximal mitochondrial oxygen consumption rate (JO₂; Panel A), mitochondrial membrane potential (Panel B), and antioxidant (AOX) buffering capacity (Panel C) and brachial artery flow-mediated dilation (FMD). Older and young adults are represented with open and closed circles, respectively. Dashed and dotted lines represent the regression lines within older and young age groups, respectively.

There was a significant relation between baseline Δψm and FMD (p = 0.01; R² = 0.20) and between maximal Δψm and FMD (p = 0.0008; R² = 0.33; Figure 5B). Additionally, there was a significant relation between Δψm and FMD at PCr2 (p = 0.03; R² = 0.15) and PCr3 (p = 0.01; R² = 0.20), but not at PCr1 (p = 0.27; R² = 0.05) (Figure S2). When examined by age group, there was a significant relation between maximal Δψm and FMD (p = 0.05; R² = 0.26) in older adults, but not in young adults (p = 0.64; R² = 0.02). There were otherwise no correlations between Δψm and FMD within either age group (all p ≥ 0.25)

There were no associations between H₂O₂ emission (p = 0.67; R² = 0.007) or production (p = 0.83; R² = 0.002) and FMD. Likewise, there was no relation between AoxBC and FMD (p = 0.11; R² = 0.10; Figure 5C). There were also no associations between H₂O₂ emission, production, or AoxBC and FMD within either age group (all p ≥ 0.23).

DISCUSSION

The primary findings from this investigation are that mitochondrial bioenergetics (JO₂ and Δψm) and AoxBC are altered in older compared with young adults. Despite lower AoxBC in older adults, we did not observe significant age differences in mtROS production or emission rates. We additionally found that altered mitochondrial bioenergetics in PBMCs – particularly Δψm and maximal JO2 – are associated with poorer endothelial function, assessed via brachial artery FMD. Collectively, the findings from this study (1) provide evidence of age-related changes in permeabilized PBMC mitochondrial bioenergetics, (2) demonstrate an association between age-related changes in PBMC mitochondrial bioenergetics and endothelial function, and (3) support the potential utility of a novel approach for the real-time assessment of mtROS production, emission, and AoxBC in PBMCs.

Few studies have examined differences in PBMC mitochondrial bioenergetics between young and typically-aging older adults. Our findings demonstrated that mitochondrial JO₂ was lower in older compared with young adults, particularly in higher energy demand states. These findings are in agreement with recent data showing poorer PBMC mitochondrial JO₂ in mostly healthy (33, 34) and frail older adults (19), and in adults with early-stage heart failure (18). On the other hand, our data contrast with one study that demonstrated only slight changes in mitochondrial respiration with aging, characterized by an age-related decline in complex I-linked respiration and a corresponding increase in complex II-linked respiration (21). The disparate findings in that study may be explained, at least in part, by the use of only a single ADP stimulus (1 mM), as opposed to a dynamic range used herein. A previous study using human muscle samples demonstrated the importance of testing mitochondrial function over a range of ADP doses (35). In that study, the magnitude of the effect of aging varied substantially across the range of ADP doses used. This further highlights the value of the CK clamp method used in the current study, assessing JO₂ over a range of ATP demand re-synthesis states.

To our knowledge, this is the first study to evaluate age-related differences in PBMC Δψm. We found that Δψm was lower (hypo-polarized) in permeabilized PBMCs from older compared with young adults across all energetic states tested. The combination of a hypo-polarized Δψm and a decreased respiratory conductance implicates at least three potential areas for further investigation into the bioenergetics changes detected in permeabilized PBMCs from older adults. First, the slight depolarization in Δψm could be due to mild uncoupling, a potential compensatory adaptation that could lessen ROS production at the expense of ATP production (36, 37). Indeed, this may help to explain the similar H₂O₂ production between young and older adults in the current study. The second involves lower abundances and/or activities of dehydrogenases within the metabolic pathways that provide NADH and FADH₂ to the mitochondria. Dehydrogenases remove electrons from carbon fuel sources (e.g., pyruvate) so that those electrons can be shuttled to the electron transport system to generate the proton motive force. A decrease in the quantity or quality of various dehydrogenases will result in less NADH/FADH₂ availability at complex-I and complex-II and could therefore lead to less proton pumping and a hypo-polarized Δψm. The third potential explanation is a loss in abundance and/or activity of the proton-pumping proteins of the electron transport system, complexes-I, III, and IV. The efficient shuttling of electrons across the electron transport system is what provides the energy to pump proton against the concentration gradient into the inner-mitochondrial space and generate a proton motive force (38). These possibilities are not necessarily mutually exclusive and our future plans are to integrate each one in the context of aging.

We used a novel assay for the assessment of real-time mtROS emission and production and AoxBC in permeabilized PBMCs. Using this assay, there were no significant age-related differences in mtROS emission and production; however, the marginal differences in emission and production between young and older adults resulted in a significant difference in AoxBC. Emitted mtROS is that which is produced and not buffered; therefore, it is arguably the most important of these variables in the context of pathophysiology. It remains unclear whether the lower AoxBC observed in older adults may translate to increased PBMC mtROS emission in response to physiological, environmental, and/or psychosocial stressors that are commonly associated with increased immune cell inflammation and oxidative stress (10, 39–41). Future work will aim to determine whether (1) age-related differences in mtROS emission and/or production become more evident in response to, for example, an inflammatory stimulus, or (2) interventions aimed toward improving mitochondrial function and reducing oxidative stress will correct age-related differences in AoxBC using this assay.

A particularly novel aspect of the current study is our investigation of the associations between PBMC mitochondrial function and endothelial function. We found that PBMC JO₂ and Δψm are associated with commonly-observed (42–45) age-related declines in brachial artery FMD. More specifically, only maximal PBMC JO₂ responses were associated with FMD, whereas Δψm was associated with FMD at all bioenergetic states with the exception of PCr1. Theoretically, altered mitochondrial bioenergetics may contribute to endothelial dysfunction by increasing oxidative stress (13, 46, 47). Notably, however, we did not observe age-related differences in mitochondrial H₂O₂ production or emission, nor did we observe any association between mitochondrial H₂O₂ production or emission and FMD responses. Alternatively, extra-mitochondrial sources of ROS (e.g., NADPH oxidase) can contribute to altered mitochondrial bioenergetics (48–50). Thus, it may be that the relations between PBMC mitochondrial bioenergetics and FMD simply reflect greater oxidative stress that is not specific to the mitochondria. Future studies may aim to better interrogate these integrated mechanisms and how they contribute to age-related changes in endothelial function.

Limitations.

The present paper represents an early step in evaluating age differences in PBMC mitochondrial function, as well as the potential impact of altered PBMC mitochondrial function on the vascular endothelium. Our data demonstrate associations between PBMC mitochondrial JO₂ and Δψm and brachial artery FMD; however, the present study is unable to provide insight with regard to causation. Additionally, our study is not adequately powered to examine potential sex differences in PBMC mitochondrial function and/or endothelial function. A previous study has demonstrated sex differences in mitochondrial function in PBMCs from older adults (51), with higher oxygen consumption rates in women compared with men. Likewise, there are sex differences in the age of onset (earlier in men) and rate (steeper in women) of vascular aging that are likely explained, at least in part, by sex hormones (42, 52). Future investigation may examine whether sex differences in PBMC mitochondrial function and vascular endothelial function are related.

Conclusions.

The present investigation evaluated age-related changes in PBMC mitochondrial bioenergetics and AoxBC. We demonstrated lower JO₂ and Δψm in older compared with young adults. Additionally, using a novel model for the real-time assessment of mtROS production and emission and AoxBC, we demonstrated lower AoxBC, despite no significant differences in mtROS production or emission. Finally, we showed that mitochondrial JO₂ and Δψm in PBMCs are related to brachial artery FMD. Collectively, these data may offer additional support for the use of PBMCs in cardiovascular research.

Supplementary Material

https://doi.org/10.6084/m9.figshare.30806948.v1