Mechanical stimuli such as shear stress and piezo1 stimulation generate red blood cell extracellular vesicles.

Introduction: Generating physiologically relevant red blood cell extracellular vesicles (RBC-EVs) for mechanistic studies is challenging. Herein, we investigated how to generate and isolate high concentrations of RBC-EVs in vitro via shear stress and mechanosensitive piezo1 ion channel stimulation. Methods: RBC-EVs were generated by applying shear stress or the piezo1-agonist yoda1 to RBCs. We then investigated how piezo1 RBC-EV generation parameters (hematocrit, treatment time, treatment dose), isolation methods (membrane-based affinity, ultrafiltration, ultracentrifugation with and without size exclusion chromatography), and storage conditions impacted RBC-EV yield and purity. Lastly, we used pressure myography to determine how RBC-EVs isolated using different methods affected mouse carotid artery vasodilation. Results: Our results showed that treating RBCs at 6% hematocrit with 10 µM yoda1 for 30 min and isolating RBC-EVs via ultracentrifugation minimized hemolysis, maximized yield and purity, and produced the most consistent RBC-EV preparations. Co-isolated contaminants in impure samples, but not piezo1 RBC-EVs, induced mouse carotid artery vasodilation. Conclusion: This work shows that RBC-EVs can be generated through piezo1 stimulation and may be generated in vivo under physiologic flow conditions. Our studies further emphasize the importance of characterizing EV generation and isolation parameters before using EVs for mechanistic analysis since RBC-EV purity can impact functional outcomes.

Circulating MicroRNA Responses to Postprandial Lipemia with or without Prior Exercise.

Repeated exposure to a high-fat meal triggers inflammation and oxidative stress, contributing to the onset of cardiometabolic diseases. Regular exercise prevents cardiometabolic diseases and a prior bout of acute endurance exercise can counteract the detrimental cardiovascular effects of a subsequent high-fat meal. Circulating microRNAs (ci-miRs) are potential mediators of these vascular effects through regulation of gene expression at the posttranscriptional level. Therefore, we investigated the expression of ci-miRs related to vascular function (miR-21, miR-92a, miR-126, miR-146a, miR-150, miR-155, miR-181b, miR-221, miR-222) in plasma from healthy, recreationally to highly active, Caucasian adult men after a high-fat meal with (EX) and without (CON) a preceding bout of cycling exercise. Ci-miR-155 was the only ci-miR for which there was a significant interaction effect of high-fat meal and exercise (p=0.050). Ci-miR-155 significantly increased in the CON group at two (p=0.007) and four hours (p=0.010) after the high-fat meal test, whereas it significantly increased in the EX group only four hours after the meal (p=0.0004). There were significant main effects of the high-fat meal on ci-miR-21 (p=0.01), ci-miR-126 (p=0.02), ci-miR-146a (p=0.02), ci-miR-181b (p=0.02), and ci-miR-221 (p=0.008). Collectively, our results suggest that prior exercise does not prevent high-fat meal-induced increases in vascular-related ci-miRs.

Race-specific changes in endothelial inflammation and microRNA in response to an acute inflammatory stimulus.

Both aberrant vascular reactivity to acute cardiovascular stress and epigenetic mechanisms such as microRNA (miR) may underlie the increased propensity for African Americans (AA) to develop cardiovascular disease. This study assessed racial differences in acute induced endothelial inflammation and related miRs. Cultured human umbilical vein endothelial cells (HUVECs) derived from AA and Caucasian Americans (CA) were exposed to influenza vaccine to determine changes in inflammatory markers, endothelial nitric oxide synthase (eNOS), and miR expression/release. Endothelial function [flow-mediated dilation (FMD)], circulating IL-6, and circulating miR were also measured in young, healthy AA and CA individuals before and after receiving the influenza vaccine. There were no significant racial differences in any parameters at baseline. The vaccine induced increases in IL-6 release (24%, P = 0.02) and ICAM-1 mRNA (40%, P = 0.03), as well as reduced eNOS mRNA (24%, P = 0.04) in AA HUVECs, but not in CA HUVECs (all P > 0.05). Intracellular levels of anti-inflammatory miR-221-3p and miR-222-3p increased specifically in CA HUVECs (72% and 53%, P = 0.04 and P = 0.06), whereas others did not change in either race. HUVEC secretion of several miRs decreased in both races, whereas the release of anti-inflammatory miR-150-5p was decreased only by AA cells (-30%, P = 0.03). In individuals of both races, circulating IL-6 increased approximately twofold 24 h after vaccination (both P < 0.01) and returned to baseline levels by 48 h, whereas FMD remained unchanged. Although macrovascular function was unaffected by acute inflammation in AA and CA individuals, AA endothelial cells exhibited increased susceptibility to acute inflammation and unique changes in related miR.NEW & NOTEWORTHY Used as an acute inflammatory stimulus, the influenza vaccine induced an inflammatory response and decreased eNOS gene expression in endothelial cells derived from African Americans, but not Caucasian Americans. Race-specific changes in intracellular expression and release of specific microRNAs also occurred and may contribute to an exaggerated inflammatory response in African Americans. In vivo, the vaccine caused similar systemic inflammation but had no effect on endothelial function or circulating microRNAs in individuals of either race.

Effects of Exercise Training on the Paracrine Function of Circulating Angiogenic Cells.

Exercise training has various benefits on cardiovascular health, and circulating angiogenic cells have been proposed as executing these changes. Work from the late 1990s supported an important role of these circulating post-natal cells in contributing to the maintenance and repair of the endothelium and vasculature. It was later found that circulating angiogenic cells were a heterogenous population of cells and primarily functioned in a paracrine manner by adhering to damaged endothelium and releasing growth factors. Many studies have discovered novel circulating angiogenic cell secreted proteins, microRNA and extracellular vesicles that mediate their angiogenic potential, and some studies have shown that both acute and chronic aerobic exercise training have distinct benefits. This review highlights work establishing an essential role of secreted factors from circulating angiogenic cells and summarizes studies regarding the effects of exercise training on these factors. Finally, we highlight the various gaps in the literature in hopes of guiding future work.

Sex-specific alterations in blood-borne factors in physically inactive individuals are detrimental to endothelial cell functions.

Mechanisms underlying the protective effects of both habitual endurance exercise and the female sex on vascular function are incompletely understood. Blood-borne circulating factors, such as circulating microRNAs (ci-miRs), may partially explain these effects. Blood samples were obtained from young, healthy men and women who either habitually performed endurance exercise (endurance trained) or were relatively inactive (sedentary). Women were tested during the early follicular phase of the menstrual cycle or the placebo pill phase of oral contraceptive to control for estrogen. Cultured human umbilical vein endothelial cells (HUVECs) were exposed to participants' serum in migration, proliferation, and reactive oxygen species (ROS) assays. Real-time quantitative polymerase chain reaction was used to quantify an initial array of 84 cardiovascular disease (CVD)-related ci-miRs, followed by validation of 10 ci-miRs. All participants were devoid of traditional CVD risk factors, and circulating estradiol concentration was not different between groups. Serum of endurance-trained women induced greater HUVEC migration compared with serum of sedentary women. HUVEC ROS production was greater in response to serum of sedentary men compared with serum of endurance-trained men and sedentary women. There were sex effects on the levels of nine ci-miRs, with greater levels in men, while ci-miRs-140-5p and 145-5p were also higher in sedentary compared with endurance-trained men and/or women. In a sex-specific manner, habitual endurance exercise was associated with beneficial effects of serum on HUVECs. Thus, alterations in circulating factors may contribute to the protective effects of habitual endurance exercise on vascular health. Additionally, sex had a greater impact than habitual activity level on the levels of vascular-related ci-miRs.NEW & NOTEWORTHY Serum from sedentary women caused impaired endothelial migration, whereas serum from sedentary men elicited increased endothelial reactive oxygen species production as compared with serum from their endurance-trained counterparts. Select CVD-related circulating microRNAs (ci-miRs) were higher in men than women, while ci-miRs-140-5p and 145-5p were also higher in sedentary versus trained men and/or women. Our data suggest that alterations in circulating factors may contribute to the protective effects of habitual exercise and sex on vascular health.

Changes in circulating microRNA and arterial stiffness following high-intensity interval and moderate intensity continuous exercise.

High-intensity interval (HII) exercise elicits distinct vascular responses compared to a matched dose of moderate intensity continuous (MOD) exercise. However, the acute effects of HII compared to MOD exercise on arterial stiffness are incompletely understood. Circulating microRNAs (ci-miRs) may contribute to the vascular effects of exercise. We sought to determine exercise intensity-dependent changes in ci-miR potentially underlying changes in arterial stiffness. Ten young, healthy men underwent well-matched, 30-min HII and MOD exercise bouts. RT-qPCR was used to determine the levels of seven vascular-related ci-miRs in serum obtained immediately before and after exercise. Arterial stiffness measures including carotid to femoral pulse wave velocity (cf-PWV), carotid arterial compliance and ß-stiffness, and augmentation index (AIx and AIx75) were taken before, 10min after and 60min after exercise. Ci-miR-21-5p, 126-3p, 126-5p, 150-5p, 155-5p, and 181b-5p increased after HII exercise (p < .05), while ci-miR-150-5p and 221-3p increased after MOD exercise (p = .03 and 0.056). One hour after HII exercise, cf-PWV trended toward being lower compared to baseline (p = .056) and was significantly lower compared to 60min after MOD exercise (p = .04). Carotid arterial compliance was increased 60min after HII exercise (p = .049) and was greater than 60min after MOD exercise (p = .02). AIx75 increased 10 min after both HII and MOD exercise (p < .05). There were significant correlations between some of the exercise-induced changes in individual ci-miRs and changes in cf-PWV and AIx/AIx75. These results support the hypotheses that arterial stiffness and ci-miRs are altered in an exercise intensity-dependent manner, and ci-miRs may contribute to changes in arterial stiffness.

The effects of moderate and high-intensity exercise on circulating markers of endothelial integrity and activation in young, healthy men.

Endothelial function typically exhibits a hormetic response to exercise. It is unknown whether endothelial damage occurs in response to acute exercise and could be a contributing mechanism. We sought to determine the effects of acute exercise on endothelial-derived circulating factors proposed to reflect endothelial integrity and activation. Young, healthy men (n = 10) underwent 30-min moderate continuous (MOD) and high-intensity interval (HII) cycling exercise bouts. Venous blood samples were taken immediately before and after exercise for quantification of circulating endothelial cells (CECs), circulating angiogenic cells (CACs), apoptotic and activated endothelial microvesicles (EMVs), thrombomodulin (TM), von Willebrand factor (vWF), syndecan-1, and circulating microRNAs (ci-miRs) 126-3p and 126-5p. Endothelial function was assessed by flow-mediated dilation (FMD) of the brachial artery before, 10 min after, and 60 min after exercise. Numbers of CECs and EMVs were unchanged by either exercise bout (P > 0.05). Numbers of all measured CAC subtypes decreased in response to MOD (21%-34%, P < 0.05), whereas only CD31+/34+/45dim/- CACs decreased following HII (21%, P < 0.05). TM and syndecan-1 increased with both exercise intensities (both ~20%, P < 0.05). HII, but not MOD, increased vWF (88%, P < 0.001), ci-miR-126-3p (92%, P = 0.009) and ci-miR-126-5p (110%, P = 0.01). The changes in several circulating factors correlated with changes in FMD following either one or both intensities. Changes in circulating factors do not support the concept of exercise-induced endothelial cell denudation, apoptosis, or activation, though slight disruption of endothelial glycocalyx and membrane integrity may occur. A related loss of mechanotransduction along with mechanisms underlying endothelial activation and ci-miR-126 secretion may relate to changes in endothelial function.NEW & NOTEWORTHY Using circulating endothelial-derived factors, we show that endothelial denudation, apoptosis, and activation do not appear to increase, whereas disrupted endothelial glycocalyx and membrane integrity may occur during both high-intensity interval and moderate intensity cycling. Increases in factors nonspecific to endothelial damage, including von Willebrand factor and microRNA-126, occurred only after high-intensity interval exercise. These results shed light on the hypothesis that disrupted endothelial integrity contributes to the endothelial function response to exercise.

Circulating microRNAs and endothelial cell migration rate are associated with metabolic syndrome and fitness level in postmenopausal African American women.

Postmenopausal African American women are at elevated risk for metabolic syndrome (MetS), which predisposes them to cardiovascular disease and other chronic diseases. Circulating microRNAs (ci-miR) are potential mediators of cardiometabolic diseases also impacted by cardiorespiratory fitness (CRF) level. Using real-time quantitative PCR, we compared the expression of vascular-related ci-miRs (miR-21-5p, miR-92a-3p, miR-126-5p, miR-146a-5p, miR-150-5p, miR-221-3p) in sedentary, overweight/obese, postmenopausal African American women based on 1) presence (n = 31) or absence (n = 42) of MetS and 2) CRF level (VO2peak ) (Very Low < 18.0 mL·kg-1 ·min-1 [n = 31], Low = 18.0-22.0 mL·kg-1 ·min-1 [n = 24], or Moderate >22.0 mL·kg-1 ·min-1 [n = 18]). Endothelial migration rate in response to subjects' serum was assessed to determine the effect of circulating blood-borne factors on endothelial repair. Ci-miR-21-5p was the only ci-miR that differed between women with MetS compared to those without MetS (0.93 ± 0.43 vs. 1.28 ± 0.71, P = 0.03). There were borderline significant differences (P = 0.06-0.09) in ci-miR-21-5p, 126-5p, and 221-3p levels between the CRF groups, and these three ci-miRs correlated with VO2peak (r = -0.25 to -0.28, P < 0.05). Endothelial migration rate was impaired in response to serum from women with MetS compared to those without after 16-24 h. Serum from women with Moderate CRF induced greater endothelial migration than the Very Low and Low CRF groups after 4 and 16-24 h, that was also not different from a young, healthy reference group. Ci-miR-21-5p is lower in postmenopausal African American women with MetS, while ci-miRs-21-5p, 126-5p, and 221-3p are associated with CRF. Factors which impair endothelial cell migration rate are present in serum of women with MetS, though having Moderate CRF may be protective.

Exercise and Cardiovascular Progenitor Cells.

Autologous stem/progenitor cell-based methods to restore blood flow and function to ischemic tissues are clinically appealing for the substantial proportion of the population with cardiovascular diseases. Early preclinical and case studies established the therapeutic potential of autologous cell therapies for neovascularization in ischemic tissues. However, trials over the past ∼15 years reveal the benefits of such therapies to be much smaller than originally estimated and a definitive clinical benefit is yet to be established. Recently, there has been an emphasis on improving the number and function of cells [herein generally referred to as circulating angiogenic cells (CACs)] used for autologous cell therapies. CACs include of several subsets of circulating cells, including endothelial progenitor cells, with proangiogenic potential that is largely exerted through paracrine functions. As exercise is known to improve CV outcomes such as angiogenesis and endothelial function, much attention is being given to exercise to improve the number and function of CACs. Accordingly, there is a growing body of evidence that acute, short-term, and chronic exercise have beneficial effects on the number and function of different subsets of CACs. In particular, recent studies show that aerobic exercise training can increase the number of CACs in circulation and enhance the function of isolated CACs as assessed in ex vivo assays. This review summarizes the roles of different subsets of CACs and the effects of acute and chronic exercise on CAC number and function, with a focus on the number and paracrine function of circulating CD34+ cells, CD31+ cells, and CD62E+ cells. © 2019 American Physiological Society. Compr Physiol 9:767-797, 2019.

Trends in aggressive play and refereeing among the top five European soccer leagues.

Current trends suggest professional soccer is becoming less aggressive, with England often argued to have the most aggressive of the top European leagues. The purpose of this study was to investigate differences in fouls and cards as indicators of aggressive play in the first divisions of England, France, Germany, Italy, and Spain over the past decade. Number of fouls per match and per yellow card has decreased in all leagues since 2007/08, though attempted tackles per foul has not changed or has increased. A lack of substantial rule changes suggests players have become less aggressive in tackling as opposed to referees becoming more lenient. Total number of fouls and cards per match were consistently lower in the English Premier League, however attempted tackles per foul was higher. The data also demonstrate the notions of home advantage and potentially referee bias, since referees tended to call more fouls and award more cards to away teams. Lastly, number of attempted tackles per foul and yellow cards received exhibited the strongest correlations with final league position across the leagues. In conclusion, our data support that elite European soccer has become less aggressive and the English Premier League is the most aggressive league.

Energy Cost of Active and Sedentary Music Video Games: Drum and Handheld Gaming vs. Walking and Sitting.

To compare energy expenditure during and after active and handheld video game drumming compared to walking and sitting. Ten experienced, college-aged men performed four protocols (one per week): no-exercise seated control (CTRL), virtual drumming on a handheld gaming device (HANDHELD), active drumming on drum pads (DRUM), and walking on a treadmill at ~30% of VO2max (WALK). Protocols were performed after an overnight fast, and expired air was collected continuously during (30min) and after (30min) exercise. DRUM and HANDHELD song lists, day of the week, and time of day were identical for each participant. Significant differences (p < 0.05) among the average rates of energy expenditure (kcal·min-1) during activity included WALK > DRUM > HANDHELD. No significant differences in the rates of energy expenditure among groups during recovery were observed. Total energy expenditure was significantly greater (p < 0.05) during WALK (149.5 ± 30.6 kcal) compared to DRUM (118.7 ± 18.8 kcal) and HANDHELD (44.9±11.6 kcal), and greater during DRUM compared to HANDHELD. Total energy expenditure was not significantly different between HANDHELD (44.9 ± 11.6 kcal) and CTRL (38.2 ± 6.0 kcal). Active video game drumming at expert-level significantly increased energy expenditure compared to handheld, but it hardly met moderate-intensity activity standards, and energy expenditure was greatest during walking. Energy expenditure with handheld video game drumming was not different from no-exercise control. Thus, traditional aerobic exercise remains at the forefront for achieving the minimum amount and intensity of physical activity for health, individuals desiring to use video games for achieving weekly physical activity recommendations should choose games that require significant involvement of lower-body musculature, and time spent playing sedentary games should be a limited part of an active lifestyle.

Laboratory and Match Physiological Data From an Elite Male Collegiate Soccer Athlete.

This study compared physiological data from an elite collegiate soccer player to those of his teammates over 2 seasons. The player of special interest (player A) was the winner of the MAC Hermann Trophy and was therefore considered the top player in National Collegiate Athletic Association (NCAA) division I soccer for each of the 2 seasons in which data were collected. Maximal oxygen consumption (V[Combining Dot Above]O2max) was measured during preseasons and heart rate (HR) was recorded during competitive matches. Polar Training Loads (PTL) were calculated using the Polar Team2 Pro (Polar USA) system based on time spent in HR zones. Player A had a lower V[Combining Dot Above]O2max than the team average in 2012 (56 vs. 61.5 ± 4.3 ml·kg·min) and a similar value in 2013 (54 vs. 56.9 ± 5.1 ml·kg·min). During matches, player A showed consistent significant differences from the team in percentage of time spent at 70-79% maximal heart rate (HRmax) (12.8 ± 5.5% vs. 10.1 ± 4.0%), 80-89% HRmax (54.3 ± 11.5% vs. 29.3 ± 6.8%), and 90-100% HRmax (23.1 ± 10.6% vs. 45.4 ± 8.5%). This led to a consistently lower PTL per minute accumulated by player A compared with his teammates (3.6 ± 0.4 vs. 4.4 ± 0.3), which may be beneficial over a season and may be related to his success. Thus, the ability to regulate moments of maximal exertion is useful in reducing training load and may be a characteristic of elite players, although whether our findings relate to differences in the playing style, position, or aerobic capacity of player A are unknown.

Investigating the extremes of the continuum of paracrine functions in CD34-/CD31+ CACs across diverse populations.

Paracrine function of circulating angiogenic cells (CACs) is thought to contribute to vascular maintenance. We previously identified S100A8 and S100A9 secreted from physically inactive individuals' CD34-/CD31+ CACs as negative regulators of capillary-like network formation. The purpose of this study was to investigate further the extremes of the continuum of CAC paracrine actions using two distinctly different groups representing "healthy" and "impaired" CAC function. We aimed to determine how capillary-like network formation in human umbilical vein endothelial cells (HUVECs) is affected by S100A8 and S100A9 in concentrations secreted by CACs from different ends of the health spectrum. CD34-/CD31+ CACs were isolated and cultured from 10 impaired function individuals defined as older (50-89 yr), non-ST-elevation myocardial infarction patients and 10 healthy individuals defined as younger (18-35 yr), healthy individuals, and conditioned media (CM) was generated. CM from the impaired function group's CACs significantly diminished network formation compared with CM from the healthy group (P < 0.05). We identified elevations in S100A8, S100A9, and S100A8/A9 in the CM from the impaired function group (P < 0.05). Pretreatment of HUVECs with inhibitors to a known S100A8 and S100A9 receptor, Toll-like receptor 4 (TLR4), but not receptor for advanced glycation end products, improved HUVEC network formation (P < 0.05) compared with CM alone in the impaired function conditions. Exposure of HUVECs to the TLR4 signaling inhibitor also blocked recombinant S100A8- and S100A9-mediated reductions in network formation. Collectively, the results suggest that the mechanisms behind impaired CAC CD34-/CD31+ CM-mediated reductions in capillary-like network formation involve secretion of S100A8 and S100A9 and binding of these proteins to TLR4 receptors on HUVECs. NEW & NOTEWORTHY: S100A8 and S100A9 proteins in concentrations secreted by CD34-/CD31+ circulating angiogenic cells (CACs) with impaired function reduce endothelial cell capillary-like network formation. These effects appear to be mediated by Toll-like receptor 4 and are absent with S100A8 and S100A9 in concentrations secreted by healthy CD34-/CD31+ CACs.

Circulating microRNAs in acute and chronic exercise: more than mere biomarkers.

MicroRNAs (miRNAs) are short, noncoding RNAs that influence biological processes by regulating gene expression after transcription. It was recently discovered that miRNAs are released into the circulation (ci-miRNAs) where they are highly stable and can act as intercellular messengers to affect physiological processes. This review provides a comprehensive summary of the studies to date that have investigated the effects of acute exercise and exercise training on ci-miRNAs in humans. Findings indicate that specific ci-miRNAs are altered in response to different protocols of acute and chronic exercise in both healthy and diseased populations. In some cases, altered ci-miRNAs correlate with fitness and health parameters, suggesting causal mechanisms by which ci-miRNAs may facilitate adaptations to exercise training. However, strong data supporting such mechanisms are lacking. Thus, a purpose of this review is to guide future studies by discussing current and novel proposed roles for ci-miRNAs in adaptations to exercise training. In addition, substantial, fundamental gaps in the field need to be addressed. The ultimate goal of this research is that an understanding of the roles of ci-miRNAs in physiological adaptations to exercise training will one day translate to therapeutic interventions.

Sex-dependent and independent effects of long-term voluntary wheel running on Bdnf mRNA and protein expression.

UNLABELLED: The beneficial effects of physical activity on brain health (synaptogenesis, neurogenesis, enhanced synaptic plasticity, improved learning and memory) appear to be mediated through changes in region-specific expression of neurotrophins, transcription factors, and postsynaptic receptors, though investigations of sex differences in response to long-term voluntary wheel running are limited. PURPOSE: To examine the effect of five months of voluntary wheel running on hippocampal mRNA and protein expression of factors critical for exercise-induced structural and functional plasticity in male and female adult mice. METHODS: At 8weeks of age, male and female C57BL/6 mice were individually housed with (PA; n=20; 10 male) or without (SED; n=20; 10 male) access to a computer monitored voluntary running wheel. At 28weeks, all mice were sacrificed and hippocampi removed. Total RNA was isolated from the hippocampus and expression of total Bdnf, Bdnf transcript IV, tPA, Pgc-1a, GluR1, NR2A, and NR2B were assessed with quantitative RT-PCR and total and mature Bdnf protein were assessed with ELISA. RESULTS: We found significantly higher Bdnf IV mRNA expression in PA males (p=0.03) and females (p=0.03) compared to SED animals. Total Bdnf mRNA expression was significantly greater in PA males compared to SED males (p=0.01), but there was no difference in females. Similarly, we observed significantly higher mature Bdnf protein in PA males compared to SED males (p=0.04), but not in females. CONCLUSION: These findings indicate that the impact of long-term voluntary wheel running on transcriptional and post-translational regulation of Bdnf may be sex-dependent, though the activity-dependent Bdnf IV transcript is sensitive to exercise independent of sex.

Chronic endurance exercise affects paracrine action of CD31+ and CD34+ cells on endothelial tube formation.

We aimed to determine if chronic endurance-exercise habits affected redox status and paracrine function of CD34(+) and CD34(-)/CD31(+) circulating angiogenic cells (CACs). Subjects were healthy, nonsmoking men and women aged 18-35 yr and categorized by chronic physical activity habits. Blood was drawn from each subject for isolation and culture of CD34(+) and CD34(-)/CD31(+) CACs. No differences in redox status were found in any group across either cell type. Conditioned media (CM) was generated from the cultured CACs and used in an in vitro human umbilical vein endothelial cell-based tube assay. CM from CD34(+) cells from inactive individuals resulted in tube structures that were 29% shorter in length (P < 0.05) and 45% less complex (P < 0.05) than the endurance-trained group. CD34(-)/CD31(+) CM from inactive subjects resulted in tube structures that were 26% shorter in length (P < 0.05) and 42% less complex (P < 0.05) than endurance-trained individuals. Proteomics analyses identified S100A8 and S100A9 in the CM. S100A9 levels were 103% higher (P < 0.05) and S100A8 was 97% higher in the CD34(-)/CD31(+) CM of inactive subjects compared with their endurance-trained counterparts with no significant differences in either protein in the CM of CD34(+) CACs as a function of training status. Recombinant S100A8/A9 treatment at concentrations detected in inactive subjects' CD34(-)/CD31(+) CAC CM also reduced tube formation (P < 0.05). These findings are the first, to our knowledge, to demonstrate a differential paracrine role in CD34(+) and CD34(-)/CD31(+) CACs on tube formation as a function of chronic physical activity habits and identifies a differential secretion of S100A9 by CD34(-)/CD31(+) CACs due to habitual exercise.