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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

Angiogenic, mitochondrial, and related transcriptional proteins were assessed in human skeletal muscle and isolated vascular cells during the early phase of endurance training. Thigh muscle biopsies were obtained in healthy young subjects, after one acute bout (n = 9) and after 3, 5, 7, and 14 days (n = 9) of cycle ergometer training. Whole muscle homogenates were analyzed for angiogenic, mitochondrial, and regulatory mRNA and protein levels. Angiogenic proteins were determined in muscle-derived endothelial cells and pericytes sorted by fluorescence-activated cell sorting. Acute exercise induced an increase in whole muscle mRNA of peroxisome proliferator-activated receptor gamma coactivator 1α (4.5-fold; P = .002) and vascular endothelial growth factor (VEGF) (2.4-fold; P = .001) at 2 hours post. After 14 days of training, there was an increase in CD31 protein (63%; P = .010) in whole muscle indicating capillary growth. There was also an increase in muscle VEGF receptor 2 (VEGFR2) (1.5-fold; P = .013), in OXPHOS proteins (complex I, II, IV, V; 1.4- to 1.9-fold; P < .05) after 14 days of training and an increase in estrogen-related receptorα protein (1.5-fold; P = .039) at 14 days compared to 5 days of training. Both endothelial cells and pericytes expressed VEGF and other angiogenic factors at the protein level but with a distinctively lower expression of VEGFR2 and thrombospondin-1 (TSP-1) in pericytes. The findings illustrate that initiation of capillary and mitochondrial adaptations occurs within 14 days of training and suggest that sustained changes in angiogenic proteins including VEGF and TSP-1 are moderate in whole muscle and vascular cells.

In human skeletal muscle, the capillary net readily adapts according to the level of muscular activity to allow for optimal diffusion conditions for oxygen from the blood to the muscle. Animal studies have demonstrated that stimulation of capillary growth in skeletal muscle can occur either by mechanical or by chemical signalling. Mechanical signals originate from shear stress forces on the endothelial cell layer induced by the blood flowing through the vessel, but include also mechanical stretch and compression of the vascular structures and the surrounding tissue, as the muscle contracts. Depending on the mechanical signal provided, capillary growth may occur either by longitudinal splitting (shear stress) or by sprouting (passive stretch). The mechanical signals initiate angiogenic processes by up-regulation or release of angioregulatory proteins that either promote, modulate or inhibit angiogenesis. A number of such regulatory proteins have been described in skeletal muscle in animal and cell models but also in human skeletal muscle. Important pro-angiogenic factors in skeletal muscle are vascular endothelial growth factor, endothelial nitric oxide synthase and angiopoietin 2, whereas angiostatic factors include thrombospondin-1 and tissue inhibitor of matrix metalloproteinase. Which of these angiogenic factors are up-regulated in the muscle tissue depends on the mechanical and chemical stimulus provided and, consequently, the process by which capillary growth occurs. The present review addresses physiological signals and angiogenic factors in skeletal muscle with a focus on human data.

Peripheral arterial disease (PAD) is caused by atherosclerosis and is associated with microcirculatory impairments in skeletal muscle. The present study evaluated the angiogenic response to exercise and passive movement in skeletal muscle of PAD patients compared with healthy control subjects. Twenty-one PAD patients and 17 aged control subjects were randomly assigned to either a passive movement or an active exercise study. Interstitial fluid microdialysate and tissue samples were obtained from the thigh skeletal muscle. Muscle dialysate vascular endothelial growth factor (VEGF) levels were modestly increased in response to either passive movement or active exercise in both subject groups. The basal muscle dialysate level of the angiostatic factor thrombospondin-1 protein was markedly higher ( P < 0.05) in PAD patients compared with the control subjects, whereas soluble VEGF receptor-1 dialysate levels were similar in the two groups. The basal VEGF protein content in the muscle tissue samples was ∼27% lower ( P < 0.05) in the PAD patients compared with the control subjects. Analysis of mRNA expression for a range of angiogenic and angiostatic factors revealed a modest change with active exercise and passive movement in both groups, except for an increase ( P < 0.05) in the ratio of angiopoietin-2 to angiopoietin-1 mRNA in the PAD group with both interventions. PAD patients and aged individuals showed a similar limited angiogenic response to active exercise and passive movement. The limited increase in muscle extracellular VEGF combined with an elevated basal level of thrombospondin-1 in muscle extracellular fluid of PAD patients may restrict capillary growth in these patients.

The subcellular distribution and secretion of vascular endothelial growth factor (VEGF) was examined in skeletal muscle of healthy humans. Skeletal muscle biopsies were obtained from m.v. lateralis before and after a 2 h bout of cycling exercise. VEGF localization was conducted on preparations of teased muscle fibers by transmission electron microscopy (TEM) and confocal microscopy (CM). Muscle interstitial fluid was sampled from microdialysis probes placed in the thigh muscle. TEM and CM analysis revealed two primary sites of localization of VEGF: in vesicles located in the subsarcolemmal regions and between the contractile elements within the muscle fibers; and in pericytes situated on the skeletal muscle capillaries. Quantitation of the subsarcolemmal density of VEGF vesicles, calculated on top of myonuclei, in the muscle fibers revealed a ∼50% increase (P

Background: Peripheral arterial disease (PAD) is characterised by impaired leg blood flow, which contributes to claudication and reduced exercise capacity. This study investigated to what extent vasoactive enzymes might contribute to altered blood flow in PAD (Fontaine stage II). Methods: We compared femoral artery blood flow during reactive hyperaemia, leg-extension exercise and passive leg movement, and determined the level of vasoactive enzymes in skeletal muscle samples from the vastus lateralis in PAD (n = 10, 68.5 ± 6.5 years) and healthy controls (CON, n = 9, 62.1 ± 12.3 years). Leg blood flow was measured with Doppler ultrasound and muscle protein levels of phosphorylated endothelial nitric oxide synthase, NADPH oxidase, cyclooxygenase 1 and 2, thromboxane synthase, and prostacyclin synthase were determined. Results: Leg blood flow during the initial 90 s of passive leg movement (242 ± 33 vs 441 ± 75 ml min-1, P = 0.03) and during reactive hyperaemia (423 ± 100 vs 1255 ± 175 ml min-1, P = 0.002) was lower in PAD than CON, whereas no significant difference was observed for leg blood flow during exercise (1490 ± 250 vs 1887 ± 349 ml min-1, P = 0.37). PAD had higher NADPH oxidase than CON (1.04 ± 0.19 vs 0.50 ± 0.06 AU, P = 0.02), with no differences for other enzymes. Leg blood flow during exercise was correlated with prostacyclin synthase (P = 0.001). Conclusion: Elevated NADPH oxidase indicates that oxidative stress may be a primary cause of low nitric oxide availability and impaired blood flow in PAD.

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.

The role of adenosine and contraction for secretion of vascular endothelial growth factor (VEGF) in skeletal muscle was investigated in human subjects and rat primary skeletal muscle cells. Microdialysis probes were inserted in the thigh muscle of seven male subjects, and dialysate was collected at rest, during infusion of adenosine, and during knee extensor exercise. The dialysate was analyzed for content of VEGF protein and adenosine. The mechanism of VEGF secretion from muscle cells in culture was examined in resting and electrostimulated cells and in response to the adenosine analog NECA and the adenosine A2A receptor specific analog CGS-21680. Adenosine receptors A1, A2A, and A2B were blocked with DPCPX, ZM-241385, and enprofylline, respectively. cAMP-dependent protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) were inhibited by H-89 and PD-98509, respectively. The human experiment showed that adenosine infusion enhanced ( P < 0.05) the interstitial concentration of VEGF protein approximately fourfold above baseline. Exercise increased ( P < 0.05) the interstitial VEGF concentration approximately sixfold above rest in parallel with an approximately threefold increase in adenosine concentration. In accordance, in cultured muscle cells, NECA and contraction caused secretion of VEGF ( P < 0.05). The contraction-induced secretion of VEGF was abolished by the A2B antagonist enprofylline and by inhibition of PKA or MAPK. The results demonstrate that adenosine causes secretion of VEGF from human skeletal muscle cells and that the contraction-induced secretion of VEGF protein is partially mediated via adenosine acting on A2B adenosine receptors. Moreover, the contraction-induced secretion of VEGF protein from muscle is dependent on both PKA and MAPK activation, but only the MAPK pathway appears to be adenosine dependent, revealing involvement of additional pathways in VEGF secretion.

The present study used passive limb movement as an experimental model to study the effect of increased blood flow and passive stretch, without enhanced metabolic demand, in young healthy male subjects. The model used was 90 min of passive movement of the leg leading to a 2.8-fold increase ( P < 0.05) in blood flow without a significant enhancement in oxygen uptake. Muscle interstitial fluid was sampled with microdialysis technique and analyzed for vascular endothelial growth factor (VEGF) protein and for the effect on endothelial cell proliferation. Biopsies obtained from the musculus vastus lateralis were analyzed for mRNA content of VEGF, endothelial nitric oxide synthase (eNOS), and matrix metalloproteinase-2 (MMP-2). The passive leg movement caused an increase ( P < 0.05) in interstitial VEGF protein concentration above rest (73 ± 21 vs. 344 ± 83 pg/ml). Addition of muscle dialysate to cultured endothelial cells revealed that dialysate obtained during leg movement induced a 3.2-fold higher proliferation rate ( P < 0.05) than dialysate obtained at rest. Passive movement also enhanced ( P < 0.05) the eNOS mRNA level fourfold above resting levels. VEGF mRNA and MMP-2 mRNA levels were unaffected. The results show that a session of passive leg movement, elevating blood flow and causing passive stretch, augments the interstitial concentrations of VEGF, the proliferative effect of interstitial fluid, and eNOS mRNA content in muscle tissue. We propose that enhanced blood flow and passive stretch are positive physiological stimulators of factors associated with capillary growth in human muscle.