Skeletal Muscle Capillarization Research Articles

Impact of exercise training on endothelial metabolism and skeletal muscle angiogenic potential in type 2 diabetes

Type 2 diabetes (T2D) is associated with vascular endothelial dysfunction and microvascular rarefaction in skeletal muscle tissue (1). Although endothelial dysfunction in metabolic disease is proposed to be associated with altered endothelial cell metabolism, there is to date a paucity of human data to support this notion. The aim of this study was to investigate the metabolism in microvascular endothelial cells derived from skeletal muscle biopsies. Moreover, as exercise training is known to reverse endothelial dysfunction and promote angiogenesis, the effect of a period of exercise training was assessed. Microvascular endothelial cells were isolated from m. vastus lateralis biopsies taken from obese individuals with (n = 7) and without (n = 8) T2D before and after 12 weeks of aerobic exercise training. Mitochondrial respiration, hydrogen peroxide (H2O2) emission, and nitric oxide (NO) formation were determined in the cells by the Oroboros Oxygraph-2K. In addition, angiogenic-, metabolic- and antioxidant enzymes and proteins, cell proliferation, and skeletal muscle capillarization were determined. At baseline, there was no difference between endothelial cells from obese control participants and T2D obese participants for any of the parameters measured: glycolytic capacity, as evidenced by a similar lactate production and PFK-1 protein content, respiratory capacity, H2O2 emission, NO emission, endothelial NO synthase protein (eNOS) content. The training period led to an overall increase in the respiratory capacity in endothelial cells from both groups (main effect, P = 0.021) whereas mitochondrial H2O2 emission remained unaltered. There was no change in glycolytic capacity, superoxide dismutase 2 or eNOS protein contents, or NO emission. Training did not influence the capillary-to-muscle fiber ratio in either group. The results of this study show that aerobic exercise training of obese control and obese diabetic subjects leads to an increase in the respiratory capacity of muscle microvascular endothelial cells. The data also indicate that there are no differences in endothelial metabolism and NO production between obese control and obese diabetic individuals. However, as the study is ongoing and only half of the subjects have been included at this time, power is limited and results may differ upon completion. Data from all subjects will be presented at the conference. Funding: The study is funded by Novo Nordisk A/S. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Making the case for resistance training in improving vascular function and skeletal muscle capillarization.

Through decades of empirical data, it has become evident that resistance training (RT) can improve strength/power and skeletal muscle hypertrophy. Yet, until recently, vascular outcomes have historically been underemphasized in RT studies, which is underscored by several exercise-related reviews supporting the benefits of endurance training on vascular measures. Several lines of evidence suggest large artery diameter and blood flow velocity increase after a single bout of resistance exercise, and these events are mediated by vasoactive substances released from endothelial cells and myofibers (e.g., nitric oxide). Weeks to months of RT can also improve basal limb blood flow and arterial diameter while lowering blood pressure. Although several older investigations suggested RT reduces skeletal muscle capillary density, this is likely due to most of these studies being cross-sectional in nature. Critically, newer evidence from longitudinal studies contradicts these findings, and a growing body of mechanistic rodent and human data suggest skeletal muscle capillarity is related to mechanical overload-induced skeletal muscle hypertrophy. In this review, we will discuss methods used by our laboratories and others to assess large artery size/function and skeletal muscle capillary characteristics. Next, we will discuss data by our groups and others examining large artery and capillary responses to a single bout of resistance exercise and chronic RT paradigms. Finally, we will discuss RT-induced mechanisms associated with acute and chronic vascular outcomes.

Physiological determinants of mechanical efficiency during advanced ageing and disuse.

This study aimed to determine which physiological factors impact net efficiency (ηnet) in oldest-old individuals at different stages of skeletal muscle disuse. To this aim, we examined ηnet, central haemodynamics, peripheral circulation, and peripheral factors (skeletal muscle fibre type, capillarization and concentration of mitochondrial DNA [mtDNA]). Twelve young (YG; 25 ± 2 years), 12 oldest-old mobile (OM; 87 ± 3 years), and 12 oldest-old immobile (OI; 88 ± 4 years) subjects performed dynamic knee extensor (KE) and elbow flexors (EF) exercise. Pulmonary oxygen uptake, photoplethysmography, Doppler ultrasound and muscle biopsies of the vastus lateralis and biceps brachii were used to assess central and peripheral adaptations to advanced ageing and disuse. Compared to the YG (12.1 ± 2.4%), the ηnet of lower-limb muscle was higher in the OM (17.6 ± 3.5%, P < 0.001), and lower in the OI (8.9 ± 1.9%, P < 0.001). These changes in ηnet during KE were coupled with significant peripheral adaptations, revealing strong correlations between ηnet and the proportion of type I muscle fibres (r=0.82), as well as [mtDNA] (r=0.77). No differences in ηnet were evident in the upper-limb muscles between YG, OM and OI. In view of the differences in limb-specific activity across the lifespan, these findings suggest that ηnet is reduced by skeletal muscle inactivity and not by chronological age, per se. Likewise, this study revealed that the age-related changes in ηnet are not a consequence of central or peripheral haemodynamic adaptations, but are likely a product of peripheral changes related to skeletal muscle fibre type and mitochondrial density. KEY POINTS: Although the effects of ageing and muscle disuse deeply impact the cardiovascular and skeletal muscle function, the combination of these factors on the mechanical efficiency are still a matter of debate. By measuring both upper- and lower-limb muscle function, which experience differing levels of disuse, we examined the influence of central and peripheral haemodynamics, and skeletal muscle factors linked to mechanical efficiency. Across the ages and degree of disuse, upper-limb muscles exhibited a preserved work economy. In the legs the oldest-old without mobility limitations exhibited an augmented mechanical efficiency, which was reduced in those with an impairment in ambulation. These changes in mechanical efficiency were associated with the proportion of type I muscle fibres. Recognition that the mechanical efficiency is not simply age-dependent, but the consequence of inactivity and subsequent skeletal muscle changes, highlights the importance of maintaining physical activity across the lifespan.

A comparison of passive heat treatment-induced vascular adaptations relative to exercise training

Epidemiological data indicate that repeated heat stress improves cardiovascular health, making passive heat therapy (PHT) a potential alternative for those unable to exercise. Few studies to date have examined the potential exercise mimetic effects in humans, and it is unclear how adaptations compare in magnitude to exercise training. OBJECTIVE: To examine the effects of 6 weeks of localized, muscle-focused PHT on resistance artery vascular function, exercise hemodynamics, and exercise performance relative to the adaptations observed following high-intensity aerobic exercise training focused on the same muscles. HYPOTHESIS: 6 weeks of PHT, applied through pulsed shortwave diathermy (2 hr, 3 days/week), would increase resistance artery function, improve exercise hemodynamics, and enhance exercise performance more than a sham treatment, but less than single-leg knee extension (KE) exercise training (EX; 40 min, 3 days/week). We also hypothesized that these functional adaptations would be accompanied by increased skeletal muscle capillarity. METHODS: We randomized 34 sedentary but otherwise healthy, young adults (ages 18–36; n = 17 female, 17 male) to receive PHT, EX, or sham heating sessions (SHAM; 2 hr, 3 days/week) over 6 weeks. Vascular function was determined through the blood flow response during both a passive leg movement (PLM) assessment and a knee extension graded exercise test (GXTmax). Muscle biopsies were taken from the vastus lateralis at baseline and after 6 weeks of intervention. RESULTS: Peak muscle treatment temperature was significantly different between all groups with PHT exhibiting a higher peak temperature (~40.80°C) than those in the EX (~37.75°C, P&lt;0.001) and SHAM groups (~36.10°C, P&lt;0.001). Peak blood flow during PLM increased to the same extent (P=0.625) in both the EX (~10.5% increase, P=0.009) and PHT groups (~8.5% increase, P=0.044); but tended to decrease in the SHAM group (P=0.087). KE peak flow increased in EX (~19%, P=0.005), but did not change in PHT (P=0.523) and decreased in SHAM (~7%, P=0.020). Peak vascular conductance during KE significantly increased by ~25% in EX (P=0.030) and PHT (P=0.012). KE peak power increased in EX by ~27% (P=0.001) but did not significantly change in PHT(P=0.175) and SHAM groups (P=0.111). EX, but not PHT or SHAM increased muscle capillary-to-fiber ratio (P = 0.0003), capillary density (P = 0.0428), and the Capillary to Fiber Perimeter Exchange Index (P = 0.0089). CONCLUSIONS: 6 weeks of localized PHT, when applied to young healthy individuals, improved resistance artery function at rest and during exercise to the same extent as exercise training. However, PHT did not lead to increased KE peak flow, microvascular remodeling, or improved exercise performance. Therefore, PHT mimics many, but not all the vascular benefits of exercise training. Further research is necessary to determine the mechanism by which 6 weeks of PHT led to improved vascular function at rest and during exercise. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Shedding light on the assessment of skeletal muscle capillarization using near-infrared spectroscopy: future directions and applications.

Shedding light on the assessment of skeletal muscle capillarization using near-infrared spectroscopy: future directions and applications.

Peroxisome proliferator-activated receptor γ coactivator 1-α overexpression improves angiogenic signalling potential of skeletal muscle-derived extracellular vesicles.

What is the central question of this study? Skeletal muscle extracellular vesicles likely act as pro-angiogenic signalling factors: does overexpression of peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) alter skeletal muscle myotube extracellular vesicle release, contents and angiogenic potential? What is the main finding and its importance? Overexpression of PGC-1α results in secretion of extracellular vesicles that elevate measures of angiogenesis and protect against acute oxidative stress in vitro. Skeletal muscle with high levels of PGC-1α expression, commonly associated with exercise induced angiogenesis and high basal capillarization, may secrete extracellular vesicles that support capillary growth and maintenance. Skeletal muscle capillarization is proportional to muscle fibre mitochondrial content and oxidative capacity. Skeletal muscle cells secrete many factors that regulate neighbouring capillary endothelial cells (ECs), including extracellular vesicles (SkM-EVs). Peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) regulates mitochondrial biogenesis and the oxidative phenotype in skeletal muscle. Skeletal muscle PGC-1α also regulates secretion of multiple angiogenic factors, but it is unknown whether PGC-1α regulates SkM-EV release, contents and angiogenic signalling potential. PGC-1α was overexpressed via adenovirus in primary human myotubes. EVs were collected from PGC-1α-overexpressing myotubes (PGC-EVs) as well as from green fluorescent protein-overexpressing myotubes (GFP-EVs), and from untreated myotubes. EV release and select mRNA contents were measured from EVs. Additionally, ECs were treated with EVs to measure angiogenic potential of EVs in normal conditions and following an oxidative stress challenge. PGC-1α overexpression did not impact EV release but did elevate EV content of mRNAs for several antioxidant proteins (nuclear factor erythroid 2-related factor 2, superoxide dismutase 2, glutathione peroxidase). PGC-EV treatment of cultured human umbilical vein endothelial cells (HUVECs) increased their proliferation (+36.6%), tube formation (length: +28.1%; number: +25.7%) and cellular viability (+52.9%), and reduced reactive oxygen species levels (-41%) compared to GFP-EVs. Additionally, PGC-EV treatment protected against tube formation impairments and induction of cellular senescence following acute oxidative stress. Overexpression of PGC-1α in human myotubes increases the angiogenic potential of SkM-EVs. These angiogenic benefits coincided with increased anti-oxidative capacity of recipient HUVECs. High PGC-1α expression in skeletal muscle may prompt the release of SkM-EVs that support vascular redox homeostasis and angiogenesis.

Angiostatic freeze or angiogenic move? Acute cold stress prevents angiokine secretion from murine myotubes but primes primary endothelial cells for greater migratory capacity.

The skeletal muscle tissue can adapt to exercise and environmental stressors with a remarkable plasticity. Prolonged cold stress exposure has been associated to increased skeletal muscle capillarization. Angioadaptation refers to the coordinated molecular and cellular processes that influence the remodeling of skeletal muscle microvasculature. Two cell types are central to angioadaptation: the myocytes, representing an important source of angiokines; and the skeletal muscle endothelial cell (SMECs), targets of these angiokines and main constituents of muscle capillaries. The influence of cold stress on skeletal muscle angioadaptation remains largely unknown, particularly with respect to myocyte-specific angiokines secretion or endothelial cell angioadaptive responses. Here, we use an in vitro model to investigate the impact of cold stress (28°C versus 37°C) on C2C12 myotubes and SMECs. Our main objectives were to evaluate: 1) the direct impact of cold stress on C2C12 cellular expression of angiokines and their release in the extracellular environment; 2) the indirect impact of cold stress on SMECs migration via these C2C12-derived angiokines; and 3) the direct effect of cold stress on SMECs angioadaptive responses, including migration, proliferation, and the activation of the vascular endothelial growth factor receptor-2 (VEGFR2). Cold stress reduced the secretion of angiokines in C2C12 myotubes culture media irrespective their pro-angiogenic or angiostatic nature. In SMECs, cold stress abrogated cell proliferation and reduced the activation of VEGFR2 despite a greater expression of this receptor. Finally, SMECs pre-conditioned to cold stress displayed an enhanced migratory response when migration was stimulated in rewarming conditions. Altogether our results suggest that cold stress may be overall angiostatic. However, cold stress accompanied by rewarming may be seen as a pro-angiogenic stressor for SMECs. This observation questions the potential for using pre-cooling in sport-performance or therapeutic exercise prescription to enhance skeletal muscle angioadaptive responses to exercise.

Short-term aerobic conditioning prior to resistance training augments muscle hypertrophy and satellite cell content in healthy young men and women.

Factors influencing inter-individual variability of responses to resistance training (RT) remain to be fully elucidated. We have proposed the importance of capillarization in skeletal muscle for the satellite cell (SC) response to RT-induced muscle hypertrophy, and hypothesized that aerobic conditioning (AC) would augment RT-induced adaptations. Fourteen healthy young (22 ± 2 years) men and women underwent AC via 6 weeks of unilateral cycling followed by 10 weeks of bilateral RT to investigate how AC alters SC content, activity, and muscle hypertrophy following RT. Muscle biopsies were taken at baseline (unilateral), post AC (bilateral), and post RT (bilateral) in the aerobically conditioned (AC + RT) and unconditioned (RT) legs. Immunofluorescence was used to determine muscle capillarization, fiber size, SC content, and activity. Type I and type II fiber cross-sectional area (CSA) increased following RT, and when legs were analyzed independently, AC + RT increased type I, type II, and mixed-fiber CSA, where the RT leg tended to increase type II (p=.05), but not type I or mixed-fiber CSA. SC content, activation, and differentiation increased with RT, where type I total and quiescent SC content was greater in AC + RT compared to the RT leg. Those with the greatest capillary-to-fiber perimeter exchange index before RT had the greatest change in CSA following RT and a significant relationship was observed between type II fiber capillarization and the change in type II-fiber CSA with RT (r=0.35). This study demonstrates that AC prior to RT can augment RT-induced muscle adaptions and that these differences are associated with increases in capillarization.

Effects of Exercise Training Intensity and Duration on Skeletal Muscle Capillarization in Healthy Subjects: A Meta-analysis.

This study aimed to investigate the effect of intensity and duration of continuous and interval exercise training on capillarization in skeletal muscle of healthy adults. PubMed and Web of Science were searched from inception to June 2021. Eligibility criteria for studies were endurance exercise training >2 wk in healthy adults, and the capillary to fiber ratio (C:F) and/or capillary density (CD) reported. Meta-analyses were performed, and subsequent subgroup analyses were conducted by the characteristics of participants and training scheme. Fifty-seven trials from 38 studies were included (10%/90%, athletic/sedentary). C:F was measured in 391 subjects from 47 trials, whereas CD was measured in 428 subjects from 50 trials. Exercise training increased C:F (mean difference, 0.33 (95% confidence interval, 0.30-0.37)) with low heterogeneity ( I2 = 45.08%) and CD (mean difference, 49.8 (36.9-62.6) capillaries per millimeter squared) with moderate heterogeneity ( I2 = 68.82%). Compared with low-intensity training (<50% of maximal oxygen consumption (V̇O 2max )), 21% higher relative change in C:F was observed after continuous moderate-intensity training (50%-80% of V̇O 2max ) and 54% higher change after interval training with high intensity (80%-100% of V̇O 2max ) in sedentary subjects. The magnitude of capillary growth was not dependent on training intervention duration. In already trained subjects, no additional increase in capillarization was observed with various types of training. In sedentary subjects, continuous moderate-intensity training and interval training with high intensity lead to increases in capillarization, whereas low-intensity training has less effect. Within the time frame studied, no effect on capillarization was established regarding training duration in sedentary subjects. The meta-analysis highlights the need for further studies in athlete groups to discern if increased capillarization can be obtained, and if so, which combination is optimal (time vs intensity).

Time course of capillary regression and an expression balance between vascular endothelial growth factor-A and thrombospondin-1 in the soleus muscle of hindlimb unloaded rats.

Skeletal muscle capillaries regress with disuse; however, information on time-dependent changes in the expression of pro- and anti-angiogenic factors in disused muscle is limited. This study aimed to clarify time-dependent changes in skeletal muscle capillarization, pro-angiogenic vascular endothelial growth factor-A (VEGF-A), and anti-angiogenic thrombospondin-1 (TSP-1) in the soleus muscle of hindlimb unloaded rat. Eight-week-old male Sprague Dawley rats were randomly divided into four groups corresponding to different hindlimb unloading (HU) duration at 0, 1, 2, and 3wk. Muscle atrophy and capillary regression worsened in the soleus muscle with longer periods of HU. The VEGF-A protein expression level was lower at week 1 than at week 0. In addition, the value at week 3 was also lower than those at weeks 0, 1, and 2. The TSP-1 protein expression level was higher at week 1 than that at week 0 but was similar at weeks 2 and 3. Moreover, reactive oxygen species, assessed by dihydroethidium fluorescence intensity on cryosection, were higher at weeks 2 and 3 than that at week 0. Depending on the HU period, VEGF-A and TSP-1 showed different expression patterns. In the early HU phase, TSP-1 may play an important role in capillary regression. However, when HU extends for a longer period, decreased VEGF-A, and/or increased oxidative stress may be more involved in capillary regression.

Exercise training in heart transplantation.

Heart transplantation remains the gold standard in the treatment of end-stage heart failure (HF). Heart transplantation patients present lower exercise capacity due to cardiovascular and musculoskeletal alterations leading thus to poor quality of life and reduction in the ability of daily self-service. Impaired vascular function and diastolic dysfunction cause lower cardiac output while decreased skeletal muscle oxidative fibers, enzymes and capillarity cause arteriovenous oxygen difference, leading thus to decreased peak oxygen uptake in heart transplant recipients. Exercise training improves exercise capacity, cardiac and vascular endothelial function in heart transplant recipients. Pre-rehabilitation regular aerobic or combined exercise is beneficial for patients with end-stage HF awaiting heart transplantation in order to maintain a higher fitness level and reduce complications afterwards like intensive care unit acquired weakness or cardiac cachexia. All hospitalized patients after heart transplantation should be referred to early mobilization of skeletal muscles through kinesiotherapy of the upper and lower limbs and respiratory physiotherapy in order to prevent infections of the respiratory system prior to hospital discharge. Moreover, all heart transplant recipients after hospital discharge who have not already participated in an early cardiac rehabilitation program should be referred to a rehabilitation center by their health care provider. Although high intensity interval training seems to have more benefits than moderate intensity continuous training, especially in stable transplant patients, individualized training based on the abilities and needs of each patient still remains the most appropriate approach. Cardiac rehabilitation appears to be safe in heart transplant patients. However, long-term follow-up data is incomplete and, therefore, further high quality and adequately-powered studies are needed to demonstrate the long-term benefits of exercise training in this population.

Effects of exercise training with weight loss on skeletal muscle expression of angiogenic factors in overweight and obese older men.

Low skeletal muscle capillarization is associated with impaired glucose tolerance (IGT); however, aerobic exercise training with weight loss (AEX + WL) increases skeletal muscle capillarization and improves glucose tolerance in adults with IGT. Given that the expression of angiogenic growth factors mediates skeletal muscle capillarization, we sought to determine whether angiogenic growth factor levels are associated with low capillarization in those with IGT versus normal glucose tolerance (NGT) or to the benefits of AEX + WL in both groups. Sixteen overweight or obese men 50-75 yr of age completed 6 mo of AEX + WL with oral glucose tolerance tests and vastus lateralis muscle biopsies for measurement of muscle vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase receptor-1 (sFlt-1), and basic fibroblast growth factor (bFGF). At baseline, all growth factor levels were numerically lower in IGT than NGT, but these did not reach statistical significance (P = 0.06-0.33). Following AEX + WL, aerobic capacity [maximal oxygen consumption (V̇o2max)] increased by 16%, whereas body weight and 120-min postprandial glucose levels decreased by 10% and 15%, respectively (P ≤ 0.001 for all). There was a main effect of AEX + WL to increase VEGF (0.095 ± 0.016 vs. 0.114 ± 0.018 ng/µg, P < 0.05), PlGF (0.004 ± 0.001 vs. 0.005 ± 0.001 ng/µg, P < 0.05), and sFlt-1 (0.216 ± 0.029 vs. 0.264 ± 0.036 ng/µg, P < 0.01), with overall increases driven by the IGT group. These results suggest that 6 mo of AEX + WL increases skeletal muscle angiogenic growth factor levels in obese older adults with IGT and NGT, which may contribute to our previous findings that AEX + WL increases capillarization to improve glucose tolerance in those with IGT.NEW & NOTEWORTHY Skeletal muscle capillarization is lower in adults with impaired glucose tolerance than normal controls. This may, in part, be attributable to differential expression of angiogenic growth factors in skeletal muscle. Using a 6-mo aerobic exercise intervention with ∼10% body weight loss (AEX + WL), we show that the expression of angiogenic growth factors tends to be lower in adults with impaired glucose tolerance compared with normal controls and that AEX + WL increased expression of angiogenic growth factors in all participants.

A High Activity Level Is Required for Augmented Muscle Capillarization in Older Women.

This study aimed to evaluate the influence of lifelong regular physical activity on skeletal muscle capillarization in women. Postmenopausal women, 61±4 yr old, were divided according to self-reported physical activity level over the past 20 yrs: sedentary (SED; n = 14), moderately active (MOD; n = 12), and very active (VERY; n = 15). Leg blood flow (LBF) was determined by ultrasound Doppler, and blood samples were drawn from the femoral artery and vein for calculation of leg oxygen uptake (LVO2) at rest and during one-legged knee extensor exercise. A skeletal muscle biopsy was obtained from the vastus lateralis and analyzed for capillarization and vascular endothelial growth factor (VEGF) and mitochondrial OXPHOS proteins. Platelets were isolated from venous blood and analyzed for VEGF content and effect on endothelial cell proliferation. The exercise-induced rise in LBF and LVO2 was faster (P = 0.008) in VERY compared with SED and MOD. Steady-state LBF and LVO2 were lower (P < 0.04) in MOD and VERY compared with SED. Capillary-fiber ratio and capillary density were greater (P < 0.03) in VERY (1.65 ± 0.48 and 409.3 ± 57.5) compared with MOD (1.30 ± 0.19 and 365.0 ± 40.2) and SED (1.30 ± 0.30 and 356.2 ± 66.3). Skeletal muscle VEGF and OXPHOS complexes I, II, and V were ~1.6-fold and ~1.25-fold (P < 0.01) higher, respectively, in VERY compared with SED. Platelets from all groups induced an approximately nine-fold (P < 0.001) increase in endothelial cell proliferation. A very active lifestyle is associated with superior skeletal muscle exercise hemodynamics and greater potential for oxygen extraction concurrent with a higher skeletal muscle capillarization and mitochondrial capacity.

Exercise intolerance in pulmonary arterial hypertension: insight into central and peripheral pathophysiological mechanisms.

Exercise intolerance is a cardinal symptom of pulmonary arterial hypertension (PAH) and strongly impacts patients' quality of life (QoL). Although central cardiopulmonary impairments limit peak oxygen consumption (V' O2peak ) in patients with PAH, several peripheral abnormalities have been described over the recent decade as key determinants in exercise intolerance, including impaired skeletal muscle (SKM) morphology, convective O2 transport, capillarity and metabolism indicating that peripheral abnormalities play a greater role in limiting exercise capacity than previously thought. More recently, cerebrovascular alterations potentially contributing to exercise intolerance in patients with PAH were also documented. Currently, only cardiopulmonary rehabilitation has been shown to efficiently improve the peripheral components of exercise intolerance in patients with PAH. However, more extensive studies are needed to identify targeted interventions that would ultimately improve patients' exercise tolerance and QoL. The present review offers a broad and comprehensive analysis of the present literature about the complex mechanisms and their interactions limiting exercise in patients and suggests several gaps in knowledge that need to be addressed in the future for a better understanding of exercise intolerance in patients with PAH.

The Impact of Lower Limb Immobilization and Rehabilitation on Angiogenic Proteins and Capillarization in Skeletal Muscle.

Skeletal muscle vascularization is important for tissue regeneration after injury and immobilization. We examined whether complete immobilization influences capillarization and oxygen delivery to the muscle and assessed the efficacy of rehabilitation by aerobic exercise training. Young healthy males had one leg immobilized for 14 d and subsequently completed 4 wk of intense aerobic exercise training. Biopsies were obtained from musculus vastus lateralis, and arteriovenous blood sampling for assessment of oxygen extraction and leg blood flow during exercise was done before and after immobilization and training. Muscle capillarization, muscle and platelet content of vascular endothelial growth factor (VEGF), and muscle thrombospondin-1 were determined. Immobilization did not have a significant impact on capillary per fiber ratio or capillary density. The content of VEGF protein in muscle samples was reduced by 36% (P = 0.024), and VEGF to thrombospondin-1 ratio was 94% lower (P = 0.046). The subsequent 4-wk training period increased the muscle VEGF content and normalized the muscle VEGF to thrombospondin-1 ratio but did not influence capillarization. Platelet VEGF content followed the trend of muscle VEGF. At the functional level, oxygen extraction, blood flow, and oxygen delivery at rest and during submaximal exercise were not affected by immobilization or training. The results demonstrate that just 2 wk of leg immobilization leads to a strongly reduced angiogenic potential as evidenced by reduced muscle and platelet VEGF content and a reduced muscle VEGF to thrombospondin-1 ratio. Moreover, a subsequent period of intensive aerobic exercise training fails to increase capillarization in the previously immobilized leg, possibly because of the angiostatic condition caused by immobilization.

Protective effects of low-intensity exercise on metabolic oxidative capacity and capillarization in skeletal muscle of non-obese diabetic rats.

Diabetes mellitus induces skeletal muscle dysfunction, such as decreased metabolic activity and capillarization. This study aimed to investigate the effects of aerobic low intensity exercise training on metabolic oxidative capacity and capillarization in skeletal muscle of non-obese diabetic rats. Eleven to twenty-five week-old male non-obese Spontaneous Diabetic Torii (SDT) rats (n = 11) and age-matched healthy male Sprague-Dawley SD rats (n = 11) were randomly assigned to either exercise or sedentary groups. The exercise training was performed on a low-speed motorized treadmill (15 m min-1) for 60 min per session, 5 sessions per week for 14 weeks in exercised groups. Sedentary SDT rats resulted in hyperglycemia, reduction of metabolic oxidative enzyme, and low percentage of oxidative fibers in the skeletal muscles. The low-intensity exercise training inhibited the growth-related increase in glucose level, and increased the muscle oxidative enzyme in exercised SDT rats compared with sedentary SDT rats. In addition, the exercise program prevented capillary regression in the skeletal muscle of diabetic rats. These results suggest that low-intensity exercise training may be an effective treatment to counter the detrimental effects of type 2 diabetes mellitus on the oxidative capacity and the capillary network of skeletal muscles.

Angiogenic potential is reduced in skeletal muscle of aged women.

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 8weeks 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. Skeletal muscle angiogenic potential was examined in cell cultures derived from aged and young women, and the effect of 8weeks 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±4.2years) and young women (n=5; 24±3.2years) 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, 8weeks 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 Impact Of CD146 +/- Serum Extracellular Vesicles On Recovery Of Skeletal Muscle Following Hindlimb Immobilization

Our laboratory recently reported a significant decline in muscle-resident pericyte quantity following hindlimb immobilization, and subsequently demonstrated the capacity for pericyte transplantation to accelerate recovery of skeletal muscle mass during the rehabilitation period. PURPOSE: The purpose of this study was to determine the extent to which pericyte-derived extracellular vesicles (EVs) effectively recover skeletal muscle mass following hindlimb immobilization. METHODS: Two groups of donor mice (n=6/group) were used to isolate serum EVs before (“Pre”) and after (“Post”) an acute bout of contraction using a sciatic nerve stimulation procedure. Serum EVs were isolated using ultracentrifugation followed by magnetic bead sorting to isolate CD146+ EVs and CD146- EVs. Five groups of mice (n=4/group) underwent unilateral hindlimb immobilization for 14 days. At 14 days, the mice were intramuscularly (tibialis anterior) injected with 1) PBS, 2) CD146+Pre EVs 3) CD146+Post EVs, 4) CD146-Pre EVs, or 5) CD146-Post EVs (in 40 μL of PBS), then remobilized for 2 weeks to determine therapeutic capacity. RESULTS: A significant decrease in serum CD146+ EVs was observed following 14 days of hindlimb immobilization (p<0.05). CD146-Post EVs demonstrated significant recovery of myofiber cross-sectional area compared to PBS control (p<0.05). CD146+Pre and CD146+Post EVs significantly restored capillary density compared to PBS control (p<0.05). CD146-Pre EVs recovered capacity for collagen remodeling compared to PBS control (p<0.05). CONCLUSIONS: The results from this study suggest that CD146- serum EVs positively benefit regrowth of skeletal muscle following a period of disuse. Additionally, CD146+ serum EVs enhance skeletal muscle capillarization. Overall, a combination of both EV fractions may optimize recovery of skeletal muscle mass following disuse. Supported by NIH NIAMS R01 AR072735 (to MDB) and ACSM NASA Space Physiology Research Grant (to SD).

Two new reliable immunohistochemical methods for simultaneous identification of capillaries, the three types of fibers and basal lamina in human skeletal muscle.

Capillary network of skeletal muscle has a crucial role in oxygen supply and is strongly associated with the phenotype and metabolic profile of muscle fibers. Abundant literature has explored capillarization of skeletal muscle in different populations and in response to different interventions. Capillary and fiber type identification techniques have considerably evolved over the last decades, but to the best of our knowledge, no validated immunohistochemical method has yet been developed to simultaneously identify capillaries (using CD31), the three different muscle fiber types, and basal lamina. Nine human muscle biopsies of vastus lateralis were stained using 5 different methods to test: the reliability of different CD31 antibodies for capillary identification, the reliability between single-section or serial-section methods, and the intra-experimenter reproducibility in visual detection of capillaries. High reliability for the different antibodies directed against capillaries was observed for capillary contacts (CC) measurements (intra-class correlations (ICC) [ICC95%] of 0.89 [0.72; 0.96] for type I fibers, 0.93 [0.81; 0.97] for type IIA fibers, 0.88 [0.71; 0.96] for type IIX fibers, 0.95 [0.86; 0.98] for all fiber types) as well as a high level of similarity between single and serial sections methods. A strong similarity in capillary analysis between the different methods was obtained for each sample measurements. Analysis of Lin's concordance correlation coefficients and Bland and Altman's graphics showed a strong intra-experimenter reproducibility. This article proposes two time- and tissue-sparing immunohistochemical methods to accurately assess a complete fiber typing (type I, IIA, and IIX) along with muscle capillarization on a single muscle section.

Long-term resveratrol treatment improves the capillarization in the skeletal muscles of ageing C57BL/6J mice

We recently showed that the treatment with Resveratrol (RES) contrasts the effects of ageing on the skeletal muscle (SKM), reduces the appearance of tubular aggregates (TAs), and improves the fatigue resistance. Since fatigue resistance depends on the SKM capillary network, and RES has been described to improve vascularisation, we analysed the SKM capillarization in naturally ageing C57BL/6J male mice, fed with 0.04% RES in the diet for 6 months, which showed a better fatigue resistance in a previous work. Our data show an inverse correlation between the number of capillaries per fibre (CAF) and TAs in both control and treated type IIB fibres, and an increase of CAF in ageing SKM upon RES-treatment. The present work suggests that capillarization is one of the determinants of the development of TAs and fatigue resistance, and that RES can be considered a good candidate to counteract capillary rarefaction in the SKM tissue.