Muscle-strain injury exudate favors acute tissue healing and prolonged connective tissue formation in humans.

Traumatic strain injury in skeletal muscle is often associated with fluid accumulation at the site of rupture, but the role of this injury exudate (EX) in cellular responses and healing is unknown. We aimed to characterize the EX sampled from human hamstring or calf muscles following a strain injury (n = 12). The cytokine and growth-factor profile, gene expression, and transcriptome analysis of EX-derived cells were compared with blood taken simultaneously from the same individuals. Cellular responses to the EX were tested in 3-dimensional (3D) culture based on primary human fibroblasts and myoblasts isolated from hamstring muscles. The EX contained a highly proinflammatory profile with a substantial expression of angiogenic factors. The proinflammatory profile was present in samples taken early postinjury and in samples aspirated several weeks postinjury, suggesting persistent inflammation. Cells derived from the EX demonstrated an increased expression of fibrogenic, adipogenic, and angiogenesis-related genes in comparison with blood cells. The injury EX stimulated fibroblast proliferation 2-fold compared with plasma, whereas such an effect was not seen for myoblasts. Finally, in 3D cell culture, the EX induced an up-regulation of connective tissue-related genes. In summary, EX formation following a muscle-strain injury stimulates fibroblast proliferation and the synthesis of connective tissue in fibroblasts. This suggests that the EX promotes an acute tissue-healing response but potentially also contributes to the formation of fibrotic tissue in the later phases of tissue repair.-Bayer, M. L., Bang, L., Hoegberget-Kalisz, M., Svensson, R. B., Olesen, J. L., Karlsson, M. M., Schjerling, P., Hellsten, Y., Hoier, B., Magnusson, S. P., Kjaer, M. Muscle-strain injury exudate favors acute tissue healing and prolonged connective tissue formation in humans.

Early time course of change in angiogenic proteins in human skeletal muscle and vascular cells with endurance training.

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.

Capillary ultrastructure and mitochondrial volume density in skeletal muscle in relation to reduced exercise capacity of patients with intermittent claudication.

Intermittent claudication (IC) is the most commonly reported symptom of peripheral arterial disease (PAD). Impaired limb blood flow is a major casual factor of lower exercise tolerance in PAD but cannot entirely explain it. We hypothesized that IC is associated with structural changes of the capillary-mitochondria interface that could contribute to the reduction of exercise tolerance in IC patients. Capillary and mitochondrial morphometry were performed after light and transmission electron microscopy using vastus lateralis muscle biopsies of 14 IC patients and 10 age-matched controls, and peak power output (PPO) was determined for all participants using an incremental single-leg knee-extension protocol. Capillary density was lower (411 ± 90 mm(-2) vs. 506 ± 95 mm(-2); P ≤ 0.05) in the biopsies of the IC patients than in those of the controls. The basement membrane (BM) around capillaries was thicker (543 ± 82 nm vs. 423 ± 97 nm; P ≤ 0.01) and the volume density of mitochondria was lower (3.51 ± 0.56% vs. 4.60 ± 0.74%; P ≤ 0.01) in the IC patients than the controls. In the IC patients, a higher proportion of capillaries appeared with collapsed slit-like lumen and/or swollen endothelium. PPO was lower (18.5 ± 9.9 W vs. 33.5 ± 9.4 W; P ≤ 0.01) in the IC patients than the controls. We suggest that several structural alterations in skeletal muscle, either collectively or separately, contribute to the reduction of exercise tolerance in IC patients.

Exercise-induced capillary growth in human skeletal muscle and the dynamics of VEGF.

In skeletal muscle, growth of capillaries is an important adaptation to exercise training that secures adequate diffusion capacity for oxygen and nutrients even at high-intensity exercise when increases in muscle blood flow are profound. Mechanical forces present during muscle activity, such as shear stress and passive stretch, lead to cellular signaling, enhanced expression of angiogenic factors, and initiation of capillary growth. The most central angiogenic factor in skeletal muscle capillary growth is VEGF. During muscle contraction, VEGF increases in the muscle interstitium, acts on VEGF receptors on the capillary endothelium, and thereby stimulates angiogenic processes. A primary source of muscle interstitial VEGF during exercise is the skeletal muscle fibers which contain large stores of VEGF within vesicles. We propose that, during muscle activity, these VEGF-containing vesicles are redistributed toward the sarcolemma where the contents are secreted into the extracellular fluid. VEGF mRNA expression is increased primarily after exercise, which allows for a more rapid replenishment of VEGF stores lost through secretion during exercise. Future studies should focus on elucidating mechanisms and regulation of VEGF secretion.

Capillary growth in human skeletal muscle: physiological factors and the balance between pro-angiogenic and angiostatic factors.

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.

Subcellular localization and mechanism of secretion of vascular endothelial growth factor in human skeletal muscle.

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<0.05) after exercise. The observation of more VEGF vesicles close to sarcolemma after exercise, combined with a 5-fold increase (P<0.05) in VEGF in the interstitial fluid, suggest that VEGF-containing vesicles redistribute to sarcolemma and that VEGF is secreted to the extracellular fluid. This study provides the first evidence in humans for a mechanism by which skeletal muscle fibers can control capillary growth by releasing VEGF from intracellular vesicles during contraction.

Aerobic High-Intensity Exercise Training Improves Cardiovascular Health in Older Post-menopausal Women.

The aim of this study was to determine the effect of a period of aerobic high intensity training on central- and peripheral cardiovascular parameters in older post-menopausal women. Eleven healthy post-menopausal (>10 years after menopause) women (mean age: 64 years; BMI: 25.3 kg m-2) completed an 8-week period of supervised, high intensity cycle training, with sessions conducted three times per week. Before and after the training period maximal oxygen uptake, body composition, popliteal artery flow mediated dilation, exercise hyperemia, arterial blood pressure, and plasma lipids were assessed. In addition, levels of estrogen related receptor α (ERRα) and vasodilator enzymes were determined in muscle biopsy samples. Training induced an 18% increase (P < 0.001) in maximal oxygen uptake. Plasma High-density lipoprotein (HDL) was higher (P < 0.05) after than before the training period. Fat mass was reduced (4.9%; P < 0.01), whereas lean body mass was unaltered. Mean arterial blood pressure was unchanged (91 vs. 88 mmHg; P = 0.058) with training. Training did not induce a change in popliteal flow mediated dilation. Exercise hyperemia at submaximal exercise was lower (P < 0.01; 11 and 4.6% at 10 and 16 W, respectively) after compared to before training. Muscle ERRα (~1.7-fold; P < 0.01) and eNOS (~1.4-fold; P < 0.05) were higher after the training intervention. The current study demonstrates that, in older post-menopausal women, a period of aerobic high intensity training effectively increases maximal oxygen uptake and improves the cardiovascular health profile, without a parallel improvement in conduit artery function.

Contraction-induced secretion of VEGF from skeletal muscle cells is mediated by adenosine.

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 A(2A) receptor specific analog CGS-21680. Adenosine receptors A(1), A(2A), and A(2B) 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 A(2B) 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 A(2B) 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.

Vasoactive enzymes and blood flow responses to passive and active exercise in peripheral arterial disease.

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.

Passive leg movement enhances interstitial VEGF protein, endothelial cell proliferation, and eNOS mRNA content in human skeletal muscle.

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.