Circulating extracellular vesicle characteristics differ between men and women following 12 weeks of concurrent exercise training.

Concurrent resistance and endurance exercise training (CET) has well-studied benefits; however, inherent hormonal and genetic differences alter adaptive responses to exercise between sexes. Extracellular vesicles (EVs) are factors that contribute to adaptive signaling. Our purpose was to test if EV characteristics differ between men and women following CET. 18 young healthy participants underwent 12-weeks of CET. Prior to and following CET, subjects performed an acute bout of heavy resistance exercise (AHRET) consisting of 6 × 10 back squats at 75% 1RM. At rest and following AHRET, EVs were isolated from plasma and characteristics and miRNA contents were analyzed. AHRET elevated EV abundance in trained men only (+51%) and AHRET-induced changes were observed for muscle-derived EVs and microvesicles. There were considerable sex-specific effects of CET on EV miRNAs, highlighted by larger variation following the 12-week program in men compared to women at rest. Pathway analysis based on differentially expressed EV miRNAs predicted that AHRET and 12 weeks of CET in men positively regulates hypertrophy and growth pathways more so than in women. This report highlights sex-based differences in the EV response to resistance and concurrent exercise training and suggests that EVs may be important adaptive signaling factors altered by exercise training.

Acute Resistance Exercise Modifies Extracellular Vesicle miRNAs Targeting Anabolic Gene Pathways: A Prospective Cohort Study.

BACKGROUND: Resistance training confers numerous health benefits that are mediated in part by circulating factors. Towards an enhanced molecular understanding, there is growing interest in a class of signaling biomarkers called extracellular vesicles (EVs). Extracellular vesicles support physiological adaptations to exercise by transporting their cargo (e.g., microRNA [miRNA]) to target cells. Previous studies of changes in EV cargo have focused on aerobic exercise, with limited data examining the effects of resistance exercise. We examined the effect of acute resistance exercise on circulating EV miRNAs and their predicted target pathways. METHODS: Ten participants (5 men; age: 26.9 ± 5.5 y, height: 1.7 ± 0.1 m, body mass: 74.0 ± 11.1 kg, body fat: 25.7 ± 11.6 %) completed an acute heavy resistance exercise test (AHRET) consisting of six sets of 10 repetitions of back squats using 75% one-repetition maximum. Pre-/post-AHRET, EVs were isolated from plasma using size exclusion chromatography, and RNA sequencing was performed. Differentially expressed (DE) miRNAs between pre- and post-AHRET EVs were analyzed using Ingenuity Pathway Analysis to predict target messenger RNAs and their target biological pathways. RESULTS: Overall, 34 miRNAs were altered by AHRET (p < 0.05), targeting 4,895 mRNAs, with enrichment of 175 canonical pathways (p < 0.01), including 12 related to growth/metabolism (p53, IGF-I, STAT3, PPAR, JAK/STAT, growth hormone, WNT/ß-catenin, ERK/MAPK, AMPK, mTOR, and PI3K/AKT) and eight to inflammation signaling (TGF-ß, IL-8, IL-7, IL-3, IL-6, IL-2, IL-17, IL-10). CONCLUSIONS: Acute resistance exercise alters EV miRNAs targeting pathways involved in growth, metabolism, and immune function. Circulating EVs may serve as significant adaptive signaling molecules influenced by exercise training.

Network-based cytokine inference implicates Oncostatin M as a driver of an inflammation phenotype in knee osteoarthritis.

Inflammatory cytokines released by synovium after trauma disturb the gene regulatory network and have been implicated in the pathophysiology of osteoarthritis. A mechanistic understanding of how aging perturbs this process can help identify novel interventions. Here, we introduced network paradigms to simulate cytokine-mediated pathological communication between the synovium and cartilage. Cartilage-specific network analysis of injured young and aged murine knees revealed aberrant matrix remodeling as a transcriptomic response unique to aged knees displaying accelerated cartilage degradation. Next, network-based cytokine inference with pharmacological manipulation uncovered IL6 family member, Oncostatin M (OSM), as a driver of the aberrant matrix remodeling. By implementing a phenotypic drug discovery approach, we identified that the activation of OSM recapitulated an "inflammatory" phenotype of knee osteoarthritis and highlighted high-value targets for drug development and repurposing. These findings offer translational opportunities targeting the inflammation-driven osteoarthritis phenotype.

Female aging: when translational models don't translate.

For many pathologies associated with aging, female patients present with higher morbidity and more frequent adverse events from treatments compared to male patients. While preclinical models are the foundation of our mechanistic understanding of age-related diseases, the most common models fail to recapitulate archetypical female aging trajectories. For example, while over 70% of the top age-related diseases are influenced by the systemic effects of reproductive senescence, we found that preclinical studies that include menopausal phenotypes modeling those seen in humans make up <1% of published aging biology research. The long-term impacts of pregnancy, birthing and breastfeeding are also typically omitted from preclinical work. In this Perspective, we summarize limitations in the most commonly used aging models, and we provide recommendations for better incorporating menopause, pregnancy and other considerations of sex in vivo and in vitro. Lastly, we outline action items for aging biology researchers, journals, funding agencies and animal providers to address this gap.

Network-based systematic dissection of exercise-induced inhibition of myosteatosis in older individuals.

Accumulated fat in skeletal muscle (i.e. myosteatosis), common in sedentary older individuals, compromises skeletal muscle health and function. A mechanistic understanding of how physical activity levels dictate fat accumulation represents a critical step towards establishment of therapies that promote healthy ageing. Using a network medicine paradigm that characterized the transcriptomic response of aged muscle to exercise versus immobilization protocols, this study explored the shared molecular cascade that regulates the fate of fibro-adipogenic progenitors (FAPs), the cell population primarily responsible for fat accumulation. Specifically, gene set enrichment analyses with network propagation revealed Pgc-1α as a functional hub of a large gene regulatory network underlying the regulation of FAPs by physical activity in aged muscle, but not in young counterparts. Integrated in silico and in situ approaches to induce Pgc-1α overexpression in aged muscle promoted mitochondrial fatty acid oxidation and inhibited FAP adipogenesis. These findings suggest that the Pgc-1α-mitochondrial fatty acid oxidation axis is a shared mechanism by which physical activity regulates age-related myosteatosis. The network medicine paradigm introduced provides mechanistic insight into exercise adaptation in elderly skeletal muscle and offers translational opportunities to advance exercise prescription for older populations. KEY POINTS: Fat accumulation is a quintessential feature of aged skeletal muscle. While increasing physical activity levels has been proposed as an effective strategy to reduce the fat in skeletal muscle (i.e. myosteatosis), the molecular cascade underlying these benefits has been poorly defined. This study implemented a series of network medicine approaches and uncovered Pgc-1α as a mechanistic driver of the regulation of fibro-adipogenic progenitors (FAPs) by physical activity. Integrated in silico and in situ approaches to induce Pgc-1α overexpression promoted mitochondrial fatty acid oxidation and inhibited FAP adipogenesis. Together, the findings of the current study suggest a novel hypothesis that physical activity reduces myosteatosis via upregulation of Pgc-1α-mediated mitochondrial fatty acid oxidation and subsequent inhibition of FAP adipogenesis.

Nanotopographical Cues Tune the Therapeutic Potential of Extracellular Vesicles for the Treatment of Aged Skeletal Muscle Injuries.

Skeletal muscle regeneration relies on the tightly temporally regulated lineage progression of muscle stem/progenitor cells (MPCs) from activation to proliferation and, finally, differentiation. However, with aging, MPC lineage progression is disrupted and delayed, ultimately causing impaired muscle regeneration. Extracellular vesicles (EVs) have attracted broad attention as next-generation therapeutics for promoting tissue regeneration. As a next step toward clinical translation, strategies to manipulate EV effects on downstream cellular targets are needed. Here, we developed an engineering strategy to tune the therapeutic potential of EVs using nanotopographical cues. We found that EVs released by young MPCs cultured on flat substrates (fEVs) promoted the proliferation of aged MPCs while EVs released by MPCs cultured on nanogratings (nEVs) promoted myogenic differentiation. We then employed a bioengineered 3D muscle aging model to optimize the administration protocol and test the therapeutic potential of fEVs and nEVs in a high-throughput manner. We found that the sequential administration first of fEVs during the phase of MPC proliferative expansion (i.e., 1 day after injury) followed by nEV administration at the stage of MPC differentiation (i.e., 3 days after injury) enhanced aged muscle regeneration to a significantly greater extent than fEVs and nEVs delivered either in isolation or mixed. The beneficial effects of the sequential EV treatment strategy were further validated in vivo, as evidenced by increased myofiber size and improved functional recovery. Collectively, our study demonstrates the ability of topographical cues to tune EV therapeutic potential and highlights the importance of optimizing the EV administration strategy to accelerate aged skeletal muscle regeneration.

Cognitive outcomes in patients treated with neuromuscular electrical stimulation after coronary artery bypass grafting.

Objective: Mechanisms of neurocognitive injury as post-operative sequelae of coronary artery bypass grafting (CABG) are not understood. The systemic inflammatory response to surgical stress causes skeletal muscle impairment, and this is also worsened by immobility. Since evidence supports a link between muscle vitality and neuroprotection, there is a need to understand the mechanisms by which promotion of muscle activity counteracts the deleterious effects of surgery on long-term cognition. Methods: We performed a clinical trial to test the hypothesis that adding neuromuscular electrical stimulation (NMES) to standard rehabilitation care in post-CABG patients promotes the maintenance of skeletal muscle strength and the expression of circulating neuroprotective myokines. Results: We did not find higher serum levels of neuroprotective myokines, except for interleukin-6, nor better long-term cognitive performance in our intervention group. However, a greater increase in functional connectivity at brain magnetic resonance was seen between seed regions within the default mode, frontoparietal, salience, and sensorimotor networks in the NMES group. Regardless of the treatment protocol, patients with a Klotho increase 3 months after hospital discharge compared to baseline Klotho values showed better scores in delayed memory tests. Significance: We confirm the potential neuroprotective effect of Klotho in a clinical setting and for the first time post-CABG.

Arsenic disrupts extracellular vesicle-mediated signaling in regenerating myofibers.

Chronic exposure to environmental arsenic is a public health crisis affecting hundreds of millions of individuals worldwide. Though arsenic is known to contribute to many pathologies and diseases, including cancers, cardiovascular and pulmonary diseases, and neurological impairment, the mechanisms for arsenic-promoted disease remain unresolved. This is especially true for arsenic impacts on skeletal muscle function and metabolism, despite the crucial role that skeletal muscle health plays in maintaining cardiovascular health, systemic homeostasis, and cognition. A barrier to researching this area is the challenge of interrogating muscle cell-specific effects in biologically relevant models. Ex vivo studies investigating mechanisms for muscle-specific responses to arsenic or other environmental contaminants primarily utilize traditional 2-dimensional culture models that cannot elucidate effects on muscle physiology or function. Therefore, we developed a contractile 3-dimensional muscle construct model-composed of primary mouse muscle progenitor cells differentiated in a hydrogel matrix-to study arsenic exposure impacts on skeletal muscle regeneration. Muscle constructs exposed to low-dose (50 nM) arsenic exhibited reduced strength and myofiber diameter following recovery from muscle injury. These effects were attributable to dysfunctional paracrine signaling mediated by extracellular vesicles (EVs) released from muscle cells. Specifically, we found that EVs collected from arsenic-exposed muscle constructs recapitulated the inhibitory effects of direct arsenic exposure on myofiber regeneration. In addition, muscle constructs treated with EVs isolated from muscles of arsenic-exposed mice displayed significantly decreased strength. Our findings highlight a novel model for muscle toxicity research and uncover a mechanism of arsenic-induced muscle dysfunction by the disruption of EV-mediated intercellular communication.

Extracellular Vesicles in Young Serum Contribute to the Restoration of Age-Related Brain Transcriptomes and Cognition in Old Mice.

We have previously demonstrated that circulating extracellular vesicles (EVs) are essential to the beneficial effect of young serum on the skeletal muscle regenerative cascade. Here, we show that infusions of young serum significantly improve age-associated memory deficits, and that these effects are abolished after serum depletion of EVs. RNA-seq analysis of the choroid plexus demonstrates EV-mediated effects on genes involved in barrier function and trans-barrier transport. Comparing the differentially expressed genes to recently published chronological aging clock genes reveals a reversal of transcriptomic aging in the choroid plexus. Following young serum treatment, the hippocampal transcriptome demonstrates significant upregulation of the anti-aging gene Klotho, along with an abrogated effect after EV depletion. Transcriptomic profiling of Klotho knockout and heterozygous mice shows the downregulation of genes associated with transport, exocytosis, and lipid transport, while upregulated genes are associated with activated microglia. The results of our study indicate the significance of EVs as vehicles to deliver signals from the periphery to the brain and the importance of Klotho in maintaining brain homeostasis.

Change in circulating klotho in response to weight loss, with and without exercise, in adults with overweight or obesity.

Introduction: Klotho is a protein associated with protection from aging-related diseases and health conditions. Obesity is associated with lower Klotho concentrations. Thus, this secondary analysis of adults with obesity examined 1) the change in serum Klotho concentration in response to a behavioral weight loss intervention by the magnitude of weight loss achieved; and 2) the association among serum Klotho concentration and weight, body composition, and cardiorespiratory fitness. Methods: Participants were randomized to either diet alone (DIET), diet plus 150 min of physical activity per week (DIET + PA150), or diet plus 250 min of physical activity per week (DIET + PA250). Participants [n = 152; age: 45.0 ± 7.9 years; body mass index (BMI): 32.4 ± 3.8 kg/m2] included in this secondary analysis provided blood samples at baseline, 6-, and 12 months, and were classified by weight loss response (Responder: achieved ≥10% weight loss at 6 or 12 months; Non-responder: achieved <5% weight loss at both 6 and 12 months). Serum Klotho was measured using a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). Analyses of covariance (ANCOVA's) were used to examine changes in weight, body composition, cardiorespiratory fitness, and Klotho concentration by weight loss response across the 12-month weight loss intervention. Results: Responders had a greater reduction in measures of weight and body composition, and a greater increase in cardiorespiratory fitness, compared to Non-Responders (p < 0.05). Change in Klotho concentration differed between Responders and Non-Responders (p < 0.05), with the increase in Klotho concentration from baseline to 6 months for Responders being statistically significant. The 6-month change in Klotho concentration was inversely associated with the 6-month change in weight (r s = -0.195), BMI (r s = -0.196), fat mass (r s = -0.184), and waist circumference (r s = -0.218) (p-values <0.05). Discussion: Findings provide evidence within the context of a behavioral intervention, with and without exercise, that change in Klotho concentration is significantly different between adults with weight loss ≥10% compared to <5% across 12 months. These findings suggest that weight loss and reduction in fat mass may be favorably associated with the change in Klotho concentration. This may reduce the risk of negative health consequences associated with accelerated aging in middle-aged adults.

Exercise-primed extracellular vesicles improve cell-matrix adhesion and chondrocyte health.

Extracellular vesicles (EVs) have been suggested to transmit the health-promoting effects of exercise throughout the body. Yet, the mechanisms by which beneficial information is transmitted from extracellular vesicles to recipient cells are poorly understood, precluding a holistic understanding of how exercise promotes cellular and tissue health. In this study, using articular cartilage as a model, we introduced a network medicine paradigm to simulate how exercise facilitates communication between circulating EVs and chondrocytes, the cells resident in articular cartilage. Using the archived small RNA-seq data of EV before and after aerobic exercise, microRNA regulatory network analysis based on network propagation inferred that circulating EVs activated by aerobic exercise perturb chondrocyte-matrix interactions and downstream cellular aging processes. Building on the mechanistic framework identified through computational analyses, follow up experimental studies interrogated the direct influence of exercise on EV-mediated chondrocyte-matrix interactions. We found that pathogenic matrix signaling in chondrocytes was abrogated in the presence of exercise-primed EVs, restoring a more youthful phenotype, as determined by chondrocyte morphological profiling and evaluation of chondrogenicity. Epigenetic reprograming of the gene encoding the longevity protein, α-Klotho, mediated these effects. These studies provide mechanistic evidence that exercise transduces rejuvenation signals to circulating EVs, endowing EVs with the capacity to ameliorate cellular health even in the presence of an unfavorable microenvironmental signals.

Neuromuscular electrical stimulation enhances the ability of serum extracellular vesicles to regenerate aged skeletal muscle after injury.

Exercise promotes healthy aging of skeletal muscle. This benefit may be mediated by youthful factors in the circulation released in response to an exercise protocol. While numerous studies to date have explored soluble proteins as systemic mediators of rejuvenating effect of exercise on tissue function, here we showed that the beneficial effect of skeletal muscle contractile activity on aged muscle function is mediated, at least in part, by regenerative properties of circulating extracellular vesicles (EVs). Muscle contractile activity elicited by neuromuscular electrical stimulation (NMES) decreased intensity of expression of the tetraspanin surface marker, CD63, on circulating EVs. Moreover, NMES shifted the biochemical Raman fingerprint of circulating EVs in aged animals with significant changes in lipid and sugar content in response to NMES when compared to controls. As a demonstration of the physiological relevance of these EV changes, we showed that intramuscular administration of EVs derived from aged animals subjected to NMES enhanced aged skeletal muscle healing after injury. These studies suggest that repetitive muscle contractile activity enhances the regenerative properties of circulating EVs in aged animals.

Dynamical modeling reveals RNA decay mediates the effect of matrix stiffness on aged muscle stem cell fate.

Loss of muscle stem cell (MuSC) self-renewal with aging reflects a combination of influences from the intracellular (e.g., post-transcriptional modifications) and extracellular (e.g., matrix stiffness) environment. Whereas conventional single cell analyses have revealed valuable insights into factors contributing to impaired self-renewal with age, most are limited by static measurements that fail to capture nonlinear dynamics. Using bioengineered matrices mimicking the stiffness of young and old muscle, we showed that while young MuSCs were unaffected by aged matrices, old MuSCs were phenotypically rejuvenated by young matrices. Dynamical modeling of RNA velocity vector fields in silico revealed that soft matrices promoted a self-renewing state in old MuSCs by attenuating RNA decay. Vector field perturbations demonstrated that the effects of matrix stiffness on MuSC self-renewal could be circumvented by fine-tuning the expression of the RNA decay machinery. These results demonstrate that post-transcriptional dynamics dictate the negative effect of aged matrices on MuSC self-renewal.

Bioengineered 3D Skeletal Muscle Model Reveals Complement 4b as a Cell-Autonomous Mechanism of Impaired Regeneration with Aging.

A mechanistic understanding of cell-autonomous skeletal muscle changes after injury can lead to novel interventions to improve functional recovery in an aged population. However, major knowledge gaps persist owing to limitations of traditional biological aging models. 2D cell culture represents an artificial environment, while aging mammalian models are contaminated by influences from non-muscle cells and other organs. Here, a 3D muscle aging system is created to overcome the limitations of these traditional platforms. It is shown that old muscle constructs (OMC) manifest a sarcopenic phenotype, as evidenced by hypotrophic myotubes, reduced contractile function, and decreased regenerative capacity compared to young muscle constructs. OMC also phenocopy the regenerative responses of aged muscle to two interventions, pharmacological and biological. Interrogation of muscle cell-specific mechanisms that contribute to impaired regeneration over time further reveals that an aging-induced increase of complement component 4b (C4b) delays muscle progenitor cell amplification and impairs functional recovery. However, administration of complement factor I, a C4b inactivator, improves muscle regeneration in vitro and in vivo, indicating that C4b inhibition may be a novel approach to enhance aged muscle repair. Collectively, the model herein exhibits capabilities to study cell-autonomous changes in skeletal muscle during aging, regeneration, and intervention.

Age-related matrix stiffening epigenetically regulates α-Klotho expression and compromises chondrocyte integrity.

Extracellular matrix stiffening is a quintessential feature of cartilage aging, a leading cause of knee osteoarthritis. Yet, the downstream molecular and cellular consequences of age-related biophysical alterations are poorly understood. Here, we show that epigenetic regulation of α-Klotho represents a novel mechanosensitive mechanism by which the aged extracellular matrix influences chondrocyte physiology. Using mass spectrometry proteomics followed by a series of genetic and pharmacological manipulations, we discovered that increased matrix stiffness drove Klotho promoter methylation, downregulated Klotho gene expression, and accelerated chondrocyte senescence in vitro. In contrast, exposing aged chondrocytes to a soft matrix restored a more youthful phenotype in vitro and enhanced cartilage integrity in vivo. Our findings demonstrate that age-related alterations in extracellular matrix biophysical properties initiate pathogenic mechanotransductive signaling that promotes Klotho promoter methylation and compromises cellular health. These findings are likely to have broad implications even beyond cartilage for the field of aging research.

Aging Affects the Efficacy of Platelet-Rich Plasma Treatment for Osteoarthritis.

OBJECTIVE: Despite the increased use of platelet-rich plasma in the treatment of osteoarthritis, whether and how age of the platelet-rich plasma donor affects therapeutic efficacy is unclear. DESIGN: In vitro, male osteoarthritic human chondrocytes were treated with platelet-rich plasma from young (18-35 yrs) or old (≥65 yrs) donors, and the chondrogenic profile was evaluated using immunofluorescent staining for two markers of chondrogenicity, type II collagen and SOX-9. In vivo, we used a within-subjects design to compare Osteoarthritis Research Society International scores in aged mouse knee joints injected with platelet-rich plasma from young or old individuals. RESULTS: In vitro experiments revealed that platelet-rich plasma from young donors induced a more youthful chondrocyte phenotype, as evidenced by increased type II collagen ( P = 0.033) and SOX-9 expression ( P = 0.022). This benefit, however, was significantly blunted when cells were cultured with platelet-rich plasma from aged donors. Accordingly, in vivo studies revealed that animals treated with platelet-rich plasma from young donors displayed a significantly improved cartilage integrity when compared with knees injected with platelet-rich plasma from aged donors ( P = 0.019). CONCLUSIONS: Injection of platelet-rich plasma from a young individual induced a regenerative effect in aged cells and mice, whereas platelet-rich plasma from aged individuals showed no improvement in chondrocyte health or cartilage integrity.

Resistance exercise differentially alters extracellular vesicle size and subpopulation characteristics in healthy men and women: an observational cohort study.

Extracellular vesicles (EVs) are established mediators of adaptation to exercise. Currently, there are no published data comparing changes in EVs between men and women after resistance exercise. We tested the hypothesis that EV profiles would demonstrate a sex-specific signature following resistance exercise. Ten men and 10 women completed an acute heavy resistance exercise test for back squats using 75% of their one-repetition maximum. Blood was drawn before and immediately after exercise. EVs were isolated from plasma using size exclusion chromatography and stained with antibodies associated with exosomes (CD63), microvesicles (VAMP3), apoptotic bodies (THSD1), and a marker for skeletal muscle EVs (SGCA). CD63+ EV concentration and proportion of total EVs increased 23% (P = 0.006) and 113% (P = 0.005) in both sexes. EV mean size declined in men (P = 0.020), but not in women, suggesting a relative increase in small EVs in men. VAMP3+ EV concentration and proportion of total EVs increased by 93% (P = 0.025) and 61% (P = 0.030) in men and women, respectively. SGCA+ EV concentration was 69% higher in women compared with men independent of time (P = 0.007). Differences were also observed for CD63, VAMP3, and SGCA median fluorescence intensity, suggesting altered surface protein density according to sex and time. There were no significant effects of time or sex on THSD1+ EVs or fluorescence intensity. EV profiles, particularly among exosome-associated and muscle-derived EVs, exhibit sex-specific differences in response to resistance exercise which should be further studied to understand their relationship to training adaptations.

Neuroendocrine, inflammatory, and extracellular vesicle responses during the Navy Special Warfare Screener Selection Course.

Military operational stress is known to increase adrenal hormones and inflammatory cytokines, while decreasing hormones associated with the anabolic milieu and neuroendocrine system. Less is known about the role of extracellular vesicles (EVs), a form of cell-to-cell communication, in military operational stress and their relationship to circulating hormones. The purpose of this study was to characterize the neuroendocrine, cytokine, and EV response to an intense. 24-h selection course known as the Naval Special Warfare (NSW) Screener and identify associations between EVs and cytokines. Blood samples were collected the morning of and following the NSW Screener in 29 men (18-26 yr). Samples were analyzed for concentrations of cortisol, insulin-like growth factor I (IGF-I), neuropeptide-Y (NPY), brain-derived neurotrophic factor (BDNF), α-klotho, tumor necrosis factor-α (TNFα), and interleukins (IL) -1ß, -6, and -10. EVs stained with markers associated with exosomes (CD63), microvesicles (VAMP3), and apoptotic bodies (THSD1) were characterized using imaging flow cytometry and vesicle flow cytometry. The selection event induced significant changes in circulating BDNF (-43.2%), IGF-I (-24.6%), TNFα (+17.7%), and IL-6 (+13.6%) accompanied by increases in intensities of THSD1+ and VAMP3+ EVs (all P < 0.05). Higher concentrations of IL-1ß and IL-10 were positively associated with THSD1+ EVs (P < 0.05). Military operational stress altered the EV profile. Surface markers associated with apoptotic bodies were positively correlated with an inflammatory response. Future studies should consider a multiomics assessment of EV cargo to discern canonical pathways that may be mediated by EVs during military stress.

Utility of extracellular vesicles as a potential biological indicator of physiological resilience during military operational stress.

Extracellular vesicles (EVs) transport biological content between cells to mediate physiological processes. The association between EVs and resilience, the ability to cope with stress, is unknown. Using unbiased machine learning approaches, we aimed to identify a biological profile of resilience. Twenty servicemen (27.8 ± 5.9 years) completed the Connor Davidson Resilience (CD-RISC) questionnaire and were exposed to daily physical and cognitive exertion with 48-hr sleep and caloric restriction. Blood samples from baseline and the second day of stress were analyzed for neuroendocrine biomarkers impacted by military stress. EVs were isolated from plasma and stained with antibodies associated with exosomes (CD63), microvesicles (VAMP3), and apoptotic bodies (THSD1). Individuals were separated into high (n = 10, CD-RISC > 90) and low (n = 10, CD-RISC < 79) resilience. EV features were stratified by size, then down-selected using regression trees and compared between groups. Diagnostic accuracy was assessed using receiver operating characteristic curves. Compared to low resilience, high resilience demonstrated a greater increase in variability of THSD1 local bright spot intensities among large-sized EVs in response to stress (p = 0.002, Hedges' g = 1.59). Among medium-sized EVs, high resilience exhibited a greater decrease in side scatter intensity (p = 0.014, Hedges' g = 1.17). Both features demonstrated high to moderate diagnostic accuracy for high resilience (AUC = 0.90 and 0.79). In contrast, neuroendocrine biomarker concentrations were similar between groups. The increase in variability among THSD1 + EVs in high, but not low, resilient individuals following stress may suggest high resilience is accompanied by stress-triggered apoptotic adaptations to the environment that are not detected in neuroendocrine biomarkers.