Mark Rakobowchuk

Human Exercise-Induced Circulating Progenitor Cell Mobilization Is Nitric Oxide-Dependent and Is Blunted in South Asian Men

Objective—Circulating progenitor cells (CPC) have emerged as potential mediators of vascular repair. In experimental models, CPC mobilization is critically dependent on nitric oxide (NO). South Asian ethnicity is associated with reduced CPC. We assessed CPC mobilization in response to exercise in Asian men and examined the role of NO in CPC mobilization per se. Methods and Results—In 15 healthy, white European men and 15 matched South Asian men, CPC mobilization was assessed during moderate-intensity exercise. Brachial artery flow-mediated vasodilatation was used to assess NO bioavailability. To determine the role of NO in CPC mobilization, identical exercise studies were performed during intravenous separate infusions of saline, the NO synthase inhibitor l-NMMA, and norepinephrine. Flow-mediated vasodilatation (5.8%±0.4% vs 7.9%±0.5%; P=0.002) and CPC mobilization (CD34+/KDR+ 53.2% vs 85.4%; P=0.001; CD133+/CD34+/KDR+ 48.4% vs 73.9%; P=0.05; and CD34+/CD45− 49.3% vs 78.4; P=0.006) was blunted in the South Asian group. CPC mobilization correlated with flow-mediated vasodilatation and l-NMMA significantly reduced exercise-induced CPC mobilization (CD34+/KDR+ −3.3% vs 68.4%; CD133+/CD34+/KDR+ 0.7% vs 71.4%; and CD34+/CD45− −30.5% vs 77.8%; all P<0.001). Conclusion—In humans, NO is critical for CPC mobilization in response to exercise. Reduced NO bioavailability may contribute to imbalance between vascular damage and repair mechanisms in South Asian men.

Heavy and moderate interval exercise training alters low-flow-mediated constriction but does not increase circulating progenitor cells in healthy humans

Moderate‐intensity endurance exercise training improves vascular endothelial vasomotor function; however, the impact of high‐intensity exercise training has been equivocal. Thus, the effect of the physiological stress of the exercise remains poorly understood. Furthermore, enhanced vascular repair mediated by circulating progenitor cells may also be improved. To address whether the physiological stress of exercise training is an important factor contributing to these adaptations, 20 healthy participants trained for 6 weeks. Training involved either moderate (MSIT; n= 9) or heavy metabolic stress (HSIT; n= 11) interval exercise training programmes matched for total work and duration of exercise. Before and after training, flow‐mediated dilatation, low‐flow‐mediated constriction and total vessel reactivity were measured at the brachial artery using Doppler ultrasound. Circulating progenitor cells (CD34+, CD133+ and CD309/KDR+) were measured by flow cytometry (means ± SD). Relative (MSIT pre‐ 5.5 ± 3.4 versus post‐training 6.6 ± 2.5%; HSIT pre‐ 6.6 ± 4.1 versus post‐training 7.0 ± 3.4%, P= 0.33) and normalized (P= 0.16) flow‐mediated dilatation did not increase with either training programme. However, low‐flow‐mediated constriction was greater after training in both groups (MSIT pre‐ −0.5 ± 3.2 versus post‐training −1.9 ± 3.1%; HSIT pre‐ −1.0 ± 1.7 versus post‐training −2.9 ± 3.0%, P= 0.04) and contributed to greater total vessel reactivity (MSIT pre‐ 7.4 ± 3.3 versus post‐training 10.1 ± 3.7%; HSIT pre‐ 10.9 ± 5.9 versus post‐training 12.7 ± 6.2%, P= 0.01). Peak reactive hyperaemia and the area under the shear rate curve were not different between groups, either before or after training. Although circulating progenitor cell numbers increased following heavy‐intensity interval exercise training, variability was great amongst participants [MSIT pre‐ 16 ± 18 versus post‐training 14 ± 12 cells (ml whole blood)−1; HSIT pre‐ 8 ± 6 versus post‐training 19 ± 23 cells (ml whole blood)−1, P= 0.50]. Overall, vasoconstrictor function may be augmented by moderate‐ and heavy‐intensity interval exercise training in young adults. However, circulating progenitor cell numbers were not increased, suggesting that these cells are not likely to be upregulated as a result of training.

Local temperature-sensitive mechanisms are important mediators of limb tissue hyperemia in the heat-stressed human at rest and during small muscle mass exercise.

Limb tissue and systemic blood flow increases with heat stress, but the underlying mechanisms remain poorly understood. Here, we tested the hypothesis that heat stress-induced increases in limb tissue perfusion are primarily mediated by local temperature-sensitive mechanisms. Leg and systemic temperatures and hemodynamics were measured at rest and during incremental single-legged knee extensor exercise in 15 males exposed to 1 h of either systemic passive heat-stress with simultaneous cooling of a single leg (n = 8) or isolated leg heating or cooling (n = 7). Systemic heat stress increased core, skin and heated leg blood temperatures (Tb), cardiac output, and heated leg blood flow (LBF; 0.6 ± 0.1 l/min; P < 0.05). In the cooled leg, however, LBF remained unchanged throughout (P > 0.05). Increased heated leg deep tissue blood flow was closely related to Tb (R2 = 0.50; P < 0.01), which is partly attributed to increases in tissue V̇O2 (R2 = 0.55; P < 0.01) accompanying elevations in total leg glucose uptake (P < 0.05). During isolated limb heating and cooling, LBFs were equivalent to those found during systemic heat stress (P > 0.05), despite unchanged systemic temperatures and hemodynamics. During incremental exercise, heated LBF was consistently maintained ∼0.6 l/min higher than that in the cooled leg (P < 0.01), with LBF and vascular conductance in both legs showing a strong correlation with their respective local Tb (R2 = 0.85 and 0.95, P < 0.05). We conclude that local temperature-sensitive mechanisms are important mediators in limb tissue perfusion regulation both at rest and during small-muscle mass exercise in hyperthermic humans.

Prolonged Low Flow Reduces Reactive Hyperemia and Augments Low Flow Mediated Constriction in the Brachial Artery Independent of the Menstrual Cycle

Non-invasive forearm ischemia-reperfusion injury and low flow induced vascular dysfunction models provide methods to evaluate vascular function. The role of oestrogen, an endogenous anti-oxidant on recovery from ischemia-reperfusion injury has not been evaluated nor has the impact of prolonged low flow on vascular function been established. Eight healthy women (33±10 yr) attended the lab during the follicular, ovulatory and mid-luteal phases of their menstrual cycles. After 30 minutes of rest, brachial artery vascular function was assessed by ultrasound measurements of diameter changes during 5 minutes of forearm ischemia and 3 minutes after. Subsequently, a 20-minute forearm ischemia period was completed. Further, vascular function assessments were completed 15, 30 and 45 minutes into recovery. Flow-mediated dilation, low-flow-mediated constriction, and reactive hyperaemia proximal to the area of ischemia were determined. Flow-mediated dilation was reduced at 15 minutes of recovery but recovered at 30 and 45 minutes (PRE: 7.1±1.0%, POST15∶4.5±0.6%, POST30∶5. 5±0.7% POST45∶5.9±0.4%, p<0.01). Conversely, low-flow mediated constriction increased (PRE: −1.3±0.4%, POST15: −3.3±0.6%, POST30: −2.5±0.5% POST45: −1.5±0.12%, p<0.01). Reactive hyperaemia was reduced throughout recovery (p<0.05). Data were unaffected by menstrual phase. Prolonged low flow altered vascular function and may relate as much to increased vasoconstriction as with decreased vasodilation. Reductions in anterograde shear and greater retrograde shear likely modulate the brachial artery response, but the reduced total shear also plays an important role. The data suggest substantial alterations in vascular function proximal to areas of ischemia with potential clinical implications following reperfusion.

Exercise intensity modulates the appearance of circulating microvesicles with pro-angiogenic potential upon endothelial cells.

The effect of endurance exercise on circulating microvesicle dynamics and their impact on surrounding endothelial cells is unclear. Here we tested the hypothesis that exercise intensity modulates the time course of platelet (PMV) and endothelial-derived (EMV) microvesicle appearance in the circulation through hemodynamic and biochemical-related mechanisms, and that microvesicles formed during exercise would stimulate endothelial angiogenesis in vitro. Nine healthy young men had venous blood samples taken before, during, and throughout the recovery period after 1 h of moderate [46 ± 2% maximal oxygen uptake (V̇o2max)] or heavy (67 ± 2% V̇o2max) intensity semirecumbent cycling and a time-matched resting control trial. In vitro experiments were performed by incubating endothelial cells with rest and exercise-derived microvesicles to examine their effects on cell angiogenic capacities. PMVs (CD41+) increased from baseline only during heavy exercise (from 21 ± 1 × 103 to 55 ± 8 × 103 and 48 ± 6 × 103 PMV/μl at 30 and 60 min, respectively; P < 0.05), returning to baseline early in postexercise recovery (P > 0.05), whereas EMVs (CD62E+) were unchanged (P > 0.05). PMVs were related to brachial artery shear rate (r2 = 0.43) and plasma norepinephrine concentrations (r2 = 0.21) during exercise (P < 0.05). Exercise-derived microvesicles enhanced endothelial proliferation, migration, and tubule formation compared with rest microvesicles (P < 0.05). These results demonstrate substantial increases in circulating PMVs during heavy exercise and that exercise-derived microvesicles stimulate human endothelial cells by enhancing angiogenesis and proliferation. This involvement of microvesicles may be considered a novel mechanism through which exercise mediates vascular healing and adaptation.

Restoring Akt1 activity in outgrowth endothelial cells from south asian men rescues vascular reparative potential

Recent data suggest reduced indices of vascular repair in South Asian men, a group at increased risk of cardiovascular events. Outgrowth endothelial cells (OEC) represent an attractive tool to study vascular repair in humans and may offer potential in cell‐based repair therapies. We aimed to define and manipulate potential mechanisms of impaired vascular repair in South Asian (SA) men. In vitro and in vivo assays of vascular repair and angiogenesis were performed using OEC derived from SA men and matched European controls, prior defining potentially causal molecular mechanisms. SA OEC exhibited impaired colony formation, migration, and in vitro angiogenesis, associated with decreased expression of the proangiogenic molecules Akt1 and endothelial nitric oxide synthase (eNOS). Transfusion of European OEC into immunodeficient mice after wire‐induced femoral artery injury augmented re‐endothelialization, in contrast with SA OEC and vehicle; SA OEC also failed to promote angiogenesis after induction of hind limb ischemia. Expression of constitutively active Akt1 (E17KAkt), but not green fluorescent protein control, in SA OEC increased in vitro angiogenesis, which was abrogated by a NOS antagonist. Moreover, E17KAkt expressing SA OEC promoted re‐endothelialization of wire‐injured femoral arteries, and perfusion recovery of ischemic limbs, to a magnitude comparable with nonmanipulated European OEC. Silencing Akt1 in European OEC recapitulated the functional deficits noted in SA OEC. Reduced signaling via the Akt/eNOS axis is causally linked with impaired OEC‐mediated vascular repair in South Asian men. These data prove the principle of rescuing marked reparative dysfunction in OEC derived from these men. Stem Cells 2014;32:2714–2723

Divergent endothelial function but similar platelet microvesicle responses following eccentric and concentric cycling at a similar aerobic power output

Endothelial function and microvesicle concentration changes after acute bouts of continuous eccentric exercise have not been assessed previously nor compared with concentric exercise at similar aerobic power outputs. This method of training may be useful among some clinical populations, but acute responses are not well described. As such, 12 healthy males completed 2 experimental sessions of either 45 min of eccentric or concentric cycling at a matched aerobic power output below the ventilatory threshold. Brachial artery vascular function was assessed throughout 5 min of forearm ischemia and 3 min thereafter, before and at 5 and 40 min of recovery following each exercise session [flow-mediated dilation (FMD)]. Venous blood samples were acquired before each vascular function assessment. FMD significantly decreased after eccentric cycling by 40 min of recovery (P < 0.05), but was unaltered after concentric exercise. No differences in peak hyperemic blood flow velocity occurred neither between modalities nor at any time point (P > 0.05). Platelet-derived microvesicles increased by ~20% after both exercise modalities (P < 0.05) while endothelial-derived microvesicles were unchanged (P > 0.05). Moderate relationships with cardiac output, a surrogate for shear stress, and norepinephrine were apparent (P < 0.05), but there were no relationships with inflammatory or acute phase proteins. In summary, eccentric endurance exercise induced macrovascular endothelial dysfunction; however, endothelial activation determined by endothelial microvesicles did not occur suggesting that this modality may induce oxidative stress but no significant endothelial damage. In addition, the increase in platelet microvesicle concentrations may induce beneficial microvascular adaptations as suggested by previous research.NEW & NOTEWORTHY Continuous eccentric cycling exercise induces substantial skeletal muscle, tendon, and bone strain providing a potentially beneficial stimulus among clinical populations. This modality also induces temporary endothelial dysfunction but no apparent damage or activation of the endothelium indicated by microvesicle production, whereas proangiogenic platelet microvesicles are released similarly following both concentric and eccentric cycling and may relate to the shear stress and catecholamine response to exercise.

Sprint Interval and Sprint Continuous Training Increases Circulating CD34 + Cells and Cardio-Respiratory Fitness in Young Healthy Women

Introduction The improvement of vascular health in the exercising limb can be attained by sprint interval training (SIT). However, the effects on systemic vascular function and on circulating angiogenic cells (CACs) which may contribute to endothelial repair have not been investigated. Additionally, a comparison between SIT and sprint continuous training (SCT) which is less time committing has not been made. Methods 12 women (22±2 yrs) completed 12 sessions of either SIT (n = 6) or work-matched SCT (n = 6) on 3 days/week. Pre and post-training assessments included brachial artery endothelial function and peripheral blood analysis for CAC number (CD34+/CD34+CD45dim). CAC function was measured by migration and adhesion assays. Cardio-respiratory fitness, carotid arterial stiffness and carotid-radial and brachial-foot pulse wave velocity (PWV) were also evaluated. Results CD34+ CACs increased following training in both groups but CD34+CD45dim did not (Pre CD34+: 40±21/105 leukocytes, Post CD34+: 56±24/105 leukocytes, main time effect p<0.05). Brachial artery flow-mediated dilation (FMD) increased following SIT but SCT had no effect (Pre SIT: 5.0±3.4%, Post SIT: 5.9±3.0%, Pre SCT: 7.2±2.7%, Post SCT: 6.5±2.9%; group x time interaction p = 0.08). increased in both training groups (Pre: 34.6±4.6 ml•kg•ml−1, Post: 36.9±5.4 ml•kg•ml−1, main time effect p<0.05). CAC function, carotid arterial stiffness and PWV did not change after training (p>0.05). Discussion SCT involving little time commitment is comparable to SIT in increasing CD34+ cell number and . An increased mobilisation of CD34+ CACs suggests that sprint training may be an effective method to enhance vascular repair.

Muscle Oxygenation Responses to Low-intensity Steady Rate Concentric and Eccentric Cycling

Muscle deoxygenation responses provide information about the training impulse of an exercise session enabling adaptation to be predicted. Our aim was to investigate muscle oxygenation profiles during prolonged low-intensity eccentric and concentric cycling. Twelve healthy men performed two 45-min exercise sessions of concentric (CON) and eccentric (ECC) cycling, matched for the same heart rate at the start of each session. Mechanical power output during ECC was ~2.5 times that of CON (210±40 W vs. 82±16 W). Oxygen uptake, blood lactate, cardiac output and systolic arterial pressure responses did not differ between exercises. Heart rate was similar at 5 min of each exercise bout but progressively increased during ECC and was higher at 15, 30 and 45 min of ECC compared to CON (+10 bpm), with a trend for a lower stroke volume. Diastolic and mean blood pressures were higher during ECC. No significant differences were observed in muscle oxygenation profiles. Muscle oxygenation responses during prolonged low-intensity exercise were not affected by the type of muscle action at the same metabolic demand and cardiac output.

Whole‐body heat stress and exercise stimulate the appearance of platelet microvesicles in plasma with limited influence of vascular shear stress

Intense, large muscle mass exercise increases circulating microvesicles, but our understanding of microvesicle dynamics and mechanisms inducing their release remains limited. However, increased vascular shear stress is generally thought to be involved. Here, we manipulated exercise‐independent and exercise‐dependent shear stress using systemic heat stress with localized single‐leg cooling (low shear) followed by single‐leg knee extensor exercise with the cooled or heated leg (Study 1, n = 8) and whole‐body passive heat stress followed by cycling (Study 2, n = 8). We quantified femoral artery shear rates (SRs) and arterial and venous platelet microvesicles (PMV–CD41+) and endothelial microvesicles (EMV–CD62E+). In Study 1, mild passive heat stress while one leg remained cooled did not affect [microvesicle] (P ≥ 0.05). Single‐leg knee extensor exercise increased active leg SRs by ~12‐fold and increased arterial and venous [PMVs] by two‐ to threefold, even in the nonexercising contralateral leg (P < 0.05). In Study 2, moderate whole‐body passive heat stress increased arterial [PMV] compared with baseline (mean±SE, from 19.9 ± 1.5 to 35.5 ± 5.4 PMV.μL−1.103, P < 0.05), and cycling with heat stress increased [PMV] further in the venous circulation (from 27.5 ± 2.2 at baseline to 57.5 ± 7.2 PMV.μL−1.103 during cycling with heat stress, P < 0.05), with a tendency for increased appearance of PMV across exercising limbs. Taken together, these findings demonstrate that whole‐body heat stress may increase arterial [PMV], and intense exercise engaging either large or small muscle mass promote PMV formation locally and systemically, with no influence upon [EMV]. Local shear stress, however, does not appear to be the major stimulus modulating PMV formation in healthy humans.