Exercise training alters skeletal muscle microvascular endothelial cell properties in recent postmenopausal females.

The present study examined and compared the impact of exercise training on redox and molecular properties of human microvascular endothelial cells derived from skeletal muscle biopsies from sedentary recent (RPF, ≤ 5 years as postmenopausal) and late (LPF, ≥ 10 years as postmenopausal) postmenopausal females. Resting skeletal muscle biopsies were obtained before and after 8 weeks of intense aerobic exercise training for isolation of microvascular endothelial cells and determination of skeletal muscle angiogenic proteins and capillarisation. The microvascular endothelial cells were analysed for mitochondrial respiration and production of reactive oxygen species (ROS), glycolysis and proteins related to vascular function, redox balance and oestrogen receptors. Exercise training led to a reduced endothelial cell ROS formation (∼50%; P = 0.009 and P = 0.020 for intact and permeabilized cells (state 3), respectively) in RPF only, with no effect on endothelial mitochondrial capacity in either group. Basal endothelial cell lactate formation was higher (7%; P = 0.028), indicating increased glycolysis, after compared to before the exercise training period in RPF only. Baseline endothelial G protein-coupled oestrogen receptor (P = 0.028) and muscle capillarisation (P = 0.028) was lower in LPF than in RPF. Muscle vascular endothelial growth factor protein was higher (32%; P = 0.002) following exercise training in LPF only. Exercise training did not influence endothelial cell proliferation or skeletal muscle capillarisation in either group, but the CD31 level in the muscle tissue, indicating endothelial cell content, was higher (>50%; P < 0.05) in both groups. In conclusion, 8 weeks of intense aerobic exercise training reduces ROS formation and enhances glycolysis in microvascular endothelial cells from RPF but does not induce skeletal muscle angiogenesis. KEY POINTS: Late postmenopausal females have been reported to achieve limited vascular adaptations to exercise training. There is a paucity of data on the effect of exercise training on isolated skeletal muscle microvascular endothelial cells (MMECs). In this study the formation of reactive oxygen species in MMECs was reduced and glycolysis increased after 8 weeks of aerobic exercise training in recent but not late postmenopausal females. Late postmenopausal females had lower levels of G protein-coupled oestrogen receptor in MMECs and lower skeletal muscle capillary density at baseline. Eight weeks of intense exercise training altered MMEC properties but did not induce skeletal muscle angiogenesis in postmenopausal females.

Exercise transiently increases the density of incipient blood clots in antiplatelet-treated lacunar stroke patients.

INTRODUCTION: Older individuals and, in particular, individuals at risk of recurrent stroke, may be susceptible to thrombosis when participating in exercise, however, this aspect has not been well investigated. METHODS: Clot microstructure and conventional markers of thrombotic risk were determined in twenty lacunar stroke patients and fifteen healthy age-matched controls before, immediately after and 1 h after a bout of moderate intensity cycling exercise. Data were analyzed using a linear mixed model approach. RESULTS: At rest, clot microstructure (1.69 ± 0.07 vs. 1.64 ± 0.05, corresponding to a difference of ~ 50% in normalized clot mass; p = 0.009) and thrombocyte count (73%; p < 0.0001) were higher, and activated partial thromboplastin time was lower (18%; p = 0.0001) in stroke patients compared to age-matched controls. Acute exercise increased thrombogenic markers similarly in the two groups: incipient clot microstructure (1.69 ± 0.07 vs. 1.74 ± 0.05; p = 0.0004 and 1.64 ± 0.05 vs. 1.71 ± 0.04; p < 0.0001, for stroke and controls respectively), plasma fibrinogen (12%; p < 0.0001 and 18%; p < 0.0001, for stroke and controls respectively) and the combined coagulation factors II, VII and X (p = 0.0001 and p < 0.0001, for stroke and controls respectively). CONCLUSION: The results show that exercise transiently increases the risk of blood clot formation in both stroke patients and controls, however, due to the higher baseline thrombogenicity in stroke patients, the post exercise risk of forming blood clots may be higher in this group. TRIAL REGISTRATION: Registered at ClinicalTrials.gov (NCT03635177).

Angiogenic potential is reduced in skeletal muscle of aged women.

KEY POINTS: The risk of cardiovascular disease and associated skeletal muscle microvascular rarefaction is enhanced in women after menopause, yet knowledge about the angiogenic potential in ageing women is generally sparse. Aged healthy and sedentary women were found to present a markedly impaired capacity for proliferation of skeletal muscle derived microvascular endothelial cells compared to young women. Vascular endothelial growth factor (VEGF) levels in skeletal muscle myocytes and release of VEGF from myocytes tended to be lower in aged compared to young women. The aged women did not show a detectable increase in skeletal muscle capillarization with 8 weeks of intense aerobic cycle training. Combined, the findings indicate that aged women have a reduced potential for capillary growth in skeletal muscle which, with ageing, may lead to age-induced microvascular rarefaction. ABSTRACT: Skeletal muscle angiogenic potential was examined in cell cultures derived from aged and young women, and the effect of 8 weeks of intense cycle training on muscle capillary growth was determined in the group of aged women. Basal muscle samples were obtained from healthy sedentary aged (n = 12; 64 ± 4.2 years) and young women (n = 5; 24 ± 3.2 years) for endothelial cell and skeletal muscle myocyte isolation and experiments. In addition, the aged women completed an 8-week training intervention. Peak oxygen uptake and muscle samples for histology and protein determination were obtained before and after the training period. Before training, muscle microdialysate was collected from the aged women at rest and during exercise. In Part 1 of the experiments, growth-supplement stimulated proliferation of endothelial cells was ∼75% lower in cells from aged compared to young women (P < 0.001). There was a tendency for a lower vascular endothelial growth factor (VEGF) concentration in muscle conditioned media (P = 0.0696) and for a lower VEGF content in the myocytes (P = 0.0705) from aged compared to young women. Endothelial proliferation was found to be highly dependent on mitochondrial function. Acute exercise resulted in a modest (1.3-fold; P = 0.0073) increase in muscle interstitial VEGF protein in the aged women. In Part 2, 8 weeks of intense training did not change muscle capillarization (P ≥ 0.1502) in the aged women, but led to an increased amount of muscle VEGF (P = 0.0339). In conclusion, aged women have impaired angiogenic potential, which is associated with a compromised response both at the skeletal muscle myocyte and microvascular endothelial cell level.

The exercise timing hypothesis: can exercise training compensate for the reduction in blood vessel function after menopause if timed right?

As women enter menopause at mid-life, oestrogen production ceases and its many beneficial effects on cardiovascular health are lost whereby the age-related risk of cardiovascular disease is accelerated. Oestrogen acts via oestrogen receptors and can activate the oestrogen response element leading to upregulation of a number of proteins of importance for vascular health, including the vasodilator and anti-atherogenic enzyme endothelial nitric oxide synthase and angiogenic factors. Hormone replacement therapy can to some extent counteract the loss of oestrogen although studies have shown that such treatment may only be effective if initiated soon after menopause, the so-called timing hypothesis. An attractive alternative to hormone therapy is regular physical activity, as it is known that exercise induces many of the same cardiovascular health protective effects as oestrogen. Nevertheless, results from studies on the effect of physical activity on vascular function and cardiovascular health are inconsistent, with some studies showing a lack of effect of a physical activity programme and others showing a beneficial effect. The reason for this divergence is unclear but here we explore whether there may be a timing aspect also for exercise training, the exercise timing hypothesis, in which initiation of exercise interventions soon after menopause may be more effective than initiation many years after. The possibility that oestrogen-related receptor-α and oxidative stress may play a role in such a timing effect is discussed.

Regulation of bone blood flow in humans: The role of nitric oxide, prostaglandins, and adenosine.

The mechanisms that regulate bone blood flow (BBF) in humans are largely unknown. Animal studies suggest that nitric oxide (NO) could be involved, and in this study, we investigated the effects of inhibition of nitric oxide synthase (NOS) alone and in combination with inhibition of cyclooxygenase (COX) enzyme, thus prostaglandin (PG) synthesis on femoral bone marrow blood flow by positron emission tomography in healthy young men at rest and during one-leg dynamic exercise. In an additional group of healthy men, the role of adenosine (ADO) in the regulation of BBF during exercise was investigated by use of an adenosine receptor blocker (aminophylline). Inhibitors were directly infused into the femoral artery. Resting BBF was 1.1 ± 0.4 mL 100 g-1 min-1 and increased to almost sixfold in response to exercise (6.3 ± 1.5 mL 100 g-1  min-1 ). Inhibition of NOS reduced BBF at rest to 0.7 ± 0.3 mL 100 g-1  min-1 (P = .036), but did not affect BBF significantly during exercise (5.5 ± 1.4 mL 100 g-1  min-1 , P = .25). On the other hand, while combined NOS and COX inhibition did not cause any further reduction of blood flow at rest (0.6 ± 0.2 mL 100 g-1 min-1 ), the combined blockade reduced BBF during exercise by ~21%, to 5.0 ± 1.8 mL 100 g-1  min-1 (P = .014). Finally, the ADO inhibition during exercise reduced BBF from 5.5 ± 1.9 mL 100 g-1  min-1 to 4.6 ± 1.2 mL 100 g-1  min-1 (P = .045). In conclusion, our results support the view that NO is involved in controlling bone marrow blood flow at rest, and NO, PG, and ADO play important roles in controlling human BBF during exercise.

Effect of endurance versus resistance training on local muscle and systemic inflammation and oxidative stress in COPD.

Limb muscle dysfunction in patients with COPD may be associated with local muscle and/or systemic inflammation, and therefore we investigated whether exercise training altered markers of inflammation and oxidative stress. We obtained vastus lateralis muscle biopsies and venous blood samples from patients with COPD (n = 30) before and after 8 weeks of resistance training (RT) (n = 15) or endurance training (ET) (n = 15). Healthy age-matched subjects were included as baseline controls (n = 8). Inflammatory markers in muscle and systemically were determined by interleukins (IL), tumour necrosis factor alfa (TNF-α), leukocyte concentration together with immunohistochemical staining for macrophages. Muscle oxidative stress and antioxidant capacity were determined by NADPH oxidase (NOX) and superoxide dismutase 2 (SOD2), respectively. Before exercise training, COPD patients had a higher muscular NOX protein content and circulating IL-8, IL-18, CRP, and leukocyte levels but a similar number of muscle-infiltrating macrophages compared with controls. Eight weeks of ET or RT increased muscle SOD2 content with no difference between groups. Plasma TNF-α, increased (P < .05) after ET and tended to (P = .06) increase after RT, but had no effect on muscular NOX protein content, number of muscle-infiltrating macrophages, or systemic levels of other pro-inflammatory cytokines or leukocytes. In patients with COPD, we found no evidence for muscular inflammation and no effect of exercise training. However, systemic inflammation was elevated in COPD and both training modalities induced an upregulation of muscle antioxidant capacity.

Vascular function in health, hypertension, and diabetes: effect of physical activity on skeletal muscle microcirculation.

Regulation of skeletal muscle blood flow is a complex process, which involves an integration of multiple mechanisms and a number of vasoactive compounds. Overall, muscle blood flow is regulated through a balance between vasoconstrictor and vasodilator signals. In a healthy cardiovascular system, the increase in muscle blood flow required for oxygen supply during exercise is achieved through a substantial increase in vasodilators locally formed in the active muscle tissue that overcome the vasoconstrictor signals. Most of the vasodilator signals are mediated via endothelial cells, which lead to the formation of vasodilators such as nitric oxide (NO) and prostacyclin. In essential hypertension and type II diabetes, the endothelial function and regulation of vascular tone is impaired with consequent increases in peripheral vascular resistance and inadequate regulation of oxygen supply to the skeletal muscle, which can affect muscle function. Central aspects in the vascular impairments are alterations in the formation of prostacyclin, the bioavailability of NO and an increased formation of vasoconstrictors and reactive oxygen species (ROS). Regular physical activity effectively improves vascular function by enhancing vasodilator formation and reducing the levels of vasoconstrictors and ROS.

The effect of purinergic P2 receptor blockade on skeletal muscle exercise hyperemia in miniature swine.

PURPOSE: ATP could play an important role in skeletal muscle blood flow regulation by inducing vasodilation via purinergic P2 receptors. This study investigated the role of P2 receptors in exercise hyperemia in miniature swine. METHODS: We measured regional blood flow with radiolabeled-microsphere technique and systemic hemodynamics before and after arterial infusion of the P2 receptor antagonist reactive blue 2 during treadmill exercise (5.2 km/h, ~60 % VO2max) and arterial ATP infusion in female Yucatan miniature swine (~29 kg). RESULTS: Mean blood flow during exercise from the 16 sampled skeletal muscle tissues was 138 ± 18 mL/min/100 g (mean ± SEM), and it was reduced in 11 (~25 %) of the 16 sampled skeletal muscles after RB2 was infused. RB2 also lowered diaphragm blood flow and kidney blood flow, whereas lung tissue blood flow was increased (all P < 0.05). Infusion of RB2 increased arterial lactate concentration during exercise from 1.6 ± 0.5 to 3.4 ± 0.6 mmol/L and heart rate from 216 ± 12 to 230 ± 9 beats/min, whereas blood pressure was unaltered. Arterial ATP infusion caused a ~twofold increase in blood flow in 15 of the 16 sampled muscle tissues and this effect was abolished after RB2 infusion. CONCLUSIONS: These results indicate that P2 receptors play a role in regulating skeletal muscle blood flow during exercise in miniature swine.

Intense intermittent exercise provides weak stimulus for vascular endothelial growth factor secretion and capillary growth in skeletal muscle.

The effect of acute intense intermittent exercise compared with moderate-intensity exercise on angiogenic factors and the effect of 4 weeks of intense intermittent training on capillary growth were examined in nine healthy young men, preconditioned by moderate-intensity endurance training. The intense training consisted of 24 bouts of 1 min cycling at an initial work rate of 316 ± 19 W (~117% of pretraining maximal oxygen uptake), performed three times per week. Skeletal muscle biopsies and muscle microdialysates were obtained from the vastus lateralis before, during and after acute exercise performed at either moderate or high intensity. Comparison of the response in angiogenic factors to acute moderate- versus high-intensity exercise, performed prior to the intense training intervention, revealed that intense exercise resulted in a markedly lower (~60%; P < 0.05) increase in interstitial vascular endothelial growth factor than did moderate-intensity exercise. Muscle interstitial fluid obtained during moderate-intensity exercise increased endothelial cell proliferation in vitro more than interstitial fluid obtained during intense exercise (sixfold versus 2.5-fold, respectively; P < 0.05). The 4 weeks of high-intensity training did not lead to an increased capillarization in the muscle but abolished the exercise-induced increase in mRNA for several angiogenic factors, increased the protein levels of endothelial nitric oxide synthase, lowered the protein levels of thrombospondin-1 in muscle but increased the interstitial protein levels of thrombospondin-1. We conclude that intense intermittent exercise provides a weak stimulus for vascular endothelial growth factor secretion and endothelial cell proliferation and that intense intermittent training does not induce a sufficient angiogenic stimulus to induce capillary growth in muscle previously conditioned by moderate-intensity exercise.

Contribution of intravascular versus interstitial purines and nitric oxide in the regulation of exercise hyperaemia in humans.

The regulation of blood flow to skeletal muscle involves a complex interaction between several locally formed vasodilators that are produced both in the skeletal muscle interstitium and intravascularly. The gas nitric oxide (NO) and the purines ATP and adenosine, are potent vasodilators that are formed by multiple cell types and released into the skeletal muscle interstitium and in plasma in response to muscle contraction. Cellular sources of ATP and NO in plasma are erythrocytes and endothelial cells, whereas interstitial sources are skeletal muscle cells and endothelial cells. Adenosine originates primarily from extracellular degradation of ATP. During exercise the concentrations of ATP and adenosine increase markedly in the interstitium with smaller increases occurring in plasma, and thus the interstitial concentration during exercise is severalfold higher than in plasma. The concentration of NO metabolites (NOx) in interstitium and plasma does not change during exercise and is similar in the two compartments. Adenosine and NO have been shown to contribute to exercise hyperaemia whereas the role of ATP remains unclear due to lack of specific purinergic receptor blockers. The relative role of intravascular versus interstitial vasodilators is not known but evidence suggests that both compartments are important. In cardiovascular disease, a reduced capacity to form adenosine in the muscle interstitium may be a contributing factor in increased peripheral vascular resistance.