Muscle Interstitial Fluid Research Articles

Mitochondrial peptides derived from exercising skeletal muscle increase oxidative stress in microvascular endothelial cells

Introduction: Mitochondria-derived peptides are newly discovered small bioactive peptides thought to communicate metabolic status to other cells. Two such peptides, MOTS-c and Humanin, have been shown to have particularly strong local and systemic regulatory effects on metabolism and oxidative stress in tissues, but their effect specifically on vascular endothelial cells remains unclear. Furthermore, it has been proposed that skeletal muscle is a source of MOTS-c and humanin during exercise but this has not been directly shown. Methods: In seven healthy young males, muscle interstitial fluid was collected from microdialysis probes in the thigh muscle, blood was drawn from femoral arterial and venous catheters and thigh muscle biopsies were obtained before and after one acute bout of exercise. In addition, muscle biopsies were obtained before and after 14 days of leg exercise training. Samples were analysed for MOTS-c, Humanin protein and mRNA. To test the effect of MOTS-c on the microvasculature, human endothelial cells derived from the muscle biopsies were incubated for three days with MOTS-c and analysed for mitochondrial respiration and formation of reactive oxygen species (ROS). Furthermore, mice were injected with MOTS-c for 4-weeks to investigate the in-vivo effect of vessels and microvascular endothelial cells. Data was analyzed by linear mixed models performed with R. Results: Acute exercise resulted in an 88% and 55% ( p<0.001) increase in MOTS-c and humanin concentration, respectively, in muscle dialysate, with no effect on the femoral arterial-venous plasma concentration differences. Both MOTS-c and humanin mRNA levels increased (by 20 and 26%; p<0.05) after acute exercise and were 43% and 35% ( p<0.05) higher, respectively, after 14 days of training. A period of MOTS-c treatment of endothelial cells increased mitochondrial respiration and ROS formation by 3-fold and 5-fold, respectively ( p<0.01). Muscle derived endothelial cells from mice injected for 4-weeks presented a similar mitochondrial respiration but a 4-fold ( p<0.05) higher ROS emission. Conclusion: interstitium in response to exercise, muscle is not a likely source of these peptides in plasma. Our data from in vitro and in vivo experiments suggest that treatment with MOTS-c does not influence endothelial metabolism but leads to increased oxidative stress in microvascular endothelial cells. The funding by Novo Nordisk Foundation (application number: 0070369) is gratefully acknowledged. 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.

Impact of charge patches on tumor disposition and biodistribution of therapeutic antibodies

This study explores the impact of antibody surface charge on tissue distribution into various tissues including tumor. Tumor-bearing mice were dosed intravenously with a mixture comprising three antibodies engineered to carry negative charge patches, a balanced charge distribution, or positive patches, respectively (cassette dosing). Tissue levels were analyzed with a specific LC-MS/MS method. In addition, the antibody mix was administered to non-tumor bearing mice. Muscle and skin interstitial fluid were obtained by centrifugation and analyzed by LC-MS/MS. An in vitro endothelium model was explored for its feasibility to mimic the observed distribution differences.A balanced charge distribution was optimal in terms of total tumor exposure, while in other tissues, negatively charged and balanced charged antibodies gave similar results. In contrast, positive charge patches generally resulted in increased serum clearance but markedly enhanced tumor and organ uptake, leading to higher tissue-to-serum ratios. The uptake and availability in the interstitial space were confirmed by specific assessment of antibody levels in the interstitial fluid of the muscle and skin, with similar charge impact as in total tissue. The in vitro model was able to differentiate the transport propensity of this series of antibody variants. In summary, our results show the differential effects of charge patches on an antibody surface on biodistribution and tumor uptake. These insights may help in the design of molecules with biodistribution properties tailored to their purpose, and an optimized safety profile.

The Colloid Osmotic Pressure Across the Glycocalyx: Role of Interstitial Fluid Sub-Compartments in Trans-Vascular Fluid Exchange in Skeletal Muscle.

The primary purpose of these investigations is to integrate our growing knowledge about the endothelial glycocalyx as a permeability and osmotic barrier into models of trans-vascular fluid exchange in whole organs. We describe changes in the colloid osmotic pressure (COP) difference for plasma proteins across the glycocalyx after an increase or decrease in capillary pressure. The composition of the fluid under the glycocalyx changes in step with capillary pressure whereas the composition of the interstitial fluid takes many hours to adjust to a change in vascular pressure. We use models where the fluid under the glycocalyx mixes with sub-compartments of the interstitial fluid (ISF) whose volumes are defined from the ultrastructure of the inter-endothelial cleft and the histology of the tissue surrounding the capillaries. The initial protein composition in the sub-compartments is that during steady state filtration in the presence of a large pore pathway in parallel with the “small pore” glycocalyx pathway. Changes in the composition depend on the volume of the sub-compartment and the balance of convective and diffusive transport into and out of each sub-compartment. In skeletal muscle the simplest model assumes that the fluid under the glycocalyx mixes directly with a tissue sub-compartment with a volume less than 20% of the total skeletal muscle interstitial fluid volume. The model places limits on trans-vascular flows during transient filtration and reabsorption over periods of 30–60 min. The key assumption in this model is compromised when the resistance to diffusion between the base of the glycocalyx and the tissue sub-compartment accounts for more than 1% of the total resistance to diffusion across the endothelial barrier. It is well established that, in the steady state, there can be no reabsorption in tissue such as skeletal muscle. Our approach extends this idea to demonstrate that transient changes in vascular pressure favoring initial reabsorption from the interstitial fluid of skeletal muscle result in much less fluid exchange than is commonly assumed. Our approach should enable critical evaluations of the empirical models of trans-vascular fluid exchange being used in the clinic that do not account for the hydrostatic and COPs across the glycocalyx.

Microdialysis-Assessed Exercised Muscle Reveals Localized and Differential IGFBP Responses to Unilateral Stretch Shortening Cycle Exercise.

Microdialysis allows for a preview into local muscle metabolism and can provide physiological insight that blood measurements cannot.Purpose: To examine the potential differential IGF-I system regulation in interstitial fluid during unilateral stretch shortening cycle exercise.Methods: 10 men (26 ± 7 year) performed unilateral jumping [stretch shortening cycle (SSC) exercise at 50% of optimal jump height] until volitional fatigue on a sled apparatus. Biological sampling took place using a catheter inserted into an antecubital vein (serum), and 100 kDa microdialysis probes inserted into the thigh muscle of each exercise/control leg (dialysate). Serum was drawn before (Pre; −3 h) and after SSC [Post I (+0 h), II (+3 h), or III (+20 h)]; dialysate was sampled for 2 h before (Pre), during/immediately after (Ex), and 3 h into recovery (Rec) following SSC. IGF-I system parameters (free/total IGF-I and IGFBPs 1–6) were measured with immunoassays. Interstitial free IGF-I was estimated from dialysate IGF-I and relative recovery (ethanol) correction. Data were analyzed with repeated measures ANOVA.Results: Serum total IGF-I remained elevated +3 h (Post II: 182.8 ± 37.6 vs. Pre: 168.3 ± 35.0 ng/mL, p < 0.01), but returned to baseline by +20 h (Post III vs. Pre, p = 0.31). No changes in serum free IGF-I were noted. Serum BP-1 and −3 increased over baseline, but not until + 20 h after SSC (Post III vs. Pre: 7.6 ± 4.9 vs. 3.7 ± 2.3 and 1,048.6 ± 269.2 vs. 891.4 ± 171.2 ng/mL, respectively). We observed a decreased serum BP-6 +3 h after SSC (p < 0.01), followed by a return to baseline at +20 h (p = 0.64 vs. Pre). There were no exercise-induced changes in serum BP-2, −4, or −5. Unlike serum, there were no changes in dialysate or interstitial free IGF-I in either leg (p > 0.05). Dialysate BP-1 remained increased in both exercise and control legs through 3 h into recovery (Rec vs. Pre, p < 0.01). Dialysate BP-3 also demonstrated a prolonged elevation over Pre SSC concentrations, but in the exercise leg only (Ex and Rec vs. Pre, p < 0.04). We observed a prolonged decrease in dialysate BP-5 (Ex and Rec vs. Pre, p < 0.03) and an increase in BP-4 IP in the exercise leg only. There were no changes relative to Pre SSC in dialysate BP-2 or −6.Conclusions: Unilateral exercise drives differential regulation of the IGF-I system at both local and systemic levels. More specifically, this is the first study to demonstrate that localized exercise increases IGFBP-3, IGFBP-4 and decreases in IGFBP-5 in muscle interstitial fluid.

Metabolomics Profiling in Response to a Cold Pressor Test

The cold pressor test (CPT) has been widely used to assess both cardiovascular and hemodynamic responses to stress. Despite the broad utilization of the CPT, there have been few if any studies in which metabolomic profiling was used to study the metabolic response to such a stimulus. Metabolomics allows insight into varying chemical processes, displaying detailed profiles of metabolites, small molecule substrates, intermediates and products of metabolism. The purpose of this study was to investigate the influence of sympathetic activation induced by a 1‐minute cold‐water immersion CPT, upon muscle metabolic responses, through metabolomics profiling of muscle interstitial fluid in adults. Five healthy adults (2 males, 3 females; 29.4 ± 9.4 yrs.; 77.6 ± 19.3 kg) were recruited for this pilot study. Three microdialysis probes (CMA 20, CMA/Microdialysis, Stockholm, Sweden) were percutaneously inserted into the vastus lateralis muscle of the dominant leg using sterile technique. Probes were then perfused with sterile saline containing 5 mM ethanol through the inlet tubing at a rate of 2.0 μL/min, and dialysate from the outlet tubing was collected in 150 μL polyethylene collection vials. One hour (3 × 20‐minute collections) of basal dialysates were collected and pooled. Participants then completed, in a seated position, a CPT in which the right hand was immersed up to the wrist in ice water for 1 minute. A 20‐min dialysate sample was collected during and immediately following the CPT. Microdialysis samples were sent to the West Coast Metabolomics Center (UC Davis Genome Center, Davis, CA, USA) for metabolomic profiling. Enzymatic‐fluorometric ethanol assay was also used to measure ethanol outflow: inflow (O:I) ratio to determine microvascular blood flow. The percent change from pre‐CPT to the CPT sample was calculated for every metabolite found. Data were then sorted on a magnitude basis, from smallest to largest change from basal levels. From the 413 metabolites detected, 207 were found to increase and 206 were found to decrease from baseline to post‐CPT. Nine metabolites were found within the 95th to 100th percentile of positive change from baseline to CPT, including: 1‐monostearin (median: 50.4%; range: 10.0–183.1), 3‐aminoisobutyric acid (median: 52.5%; range: 39.2–95.4), palmitoleic acid (median: 92.2%; range: 35.4–204.5); and 6 “unknown metabolites.” Furthermore, 5 “unknown metabolites” were also detected within the 95th to 100th percentile of negative change (decrease from baseline). Ethanol O:I ratio increased baseline to CPT, indicating decreased blood flow (0.483 ± 0.088 to 0.547 ± 0.027, mean ± SEM). These results indicate that the CPT can elicit both vasoconstriction and a notable metabolic response in muscle tissue, identifiable by the use of ethanol analysis and metabolomics of dialysate from microdialysis of skeletal muscle. Of specific interest, 1‐monostearin and palmitoleic acid were found to be in the group of molecules that increased the most in response to CPT. Although speculative, increased adrenergic stimulation resulting from CPT‐induced sympathetic activation may have resulted in liberation of 1‐monostearin and palmitoleic acid from membranes or other locations, or from increases in desaturase activities.

Skeletal Muscle Stretch‐Induced PGE2 Formation does not Impact the Muscle Mechanoreflex in Decerebrate Rats

Mechanical and metabolic signals associated with skeletal muscle contraction stimulate thin fiber muscle afferents and produce reflex increases in blood pressure (i.e., the exercise pressor reflex). There is an interplay between those signals as metabolites may sensitize afferent responsiveness to mechanical signals. A common strategy used to study the mechanical component of the exercise pressor reflex (i.e., the mechanoreflex) in isolation from the metabolic component is to measure the reflex increase in blood pressure during passive hindlimb muscle stretch in an experimental animal preparation. Passive muscle stretch has been presumed to impart a purely mechanical stimulus. However, intermittent but not static mechanical stimulation of cultured skeletal muscle cells was found to increase PGE2 production and PGE2 has been shown to sensitize the muscle mechanoreflex. Based on those findings, the purpose of this investigation was to test the hypothesis that in decerebrate, unanesthetized rats, dynamic but not static hindlimb skeletal muscle stretch would increase interstitial PGE2 concentration. Moreover, we tested the hypothesis that COX inhibition with indomethacin would attenuate the stretch‐induced increase in PGE2 concentration and the increase in blood pressure during mechanoreflex activation. Experiments were performed on 28 young adult male Sprague‐Dawley rats. The microdialysis technique was used to collect triceps surae muscle interstitial fluid at rest and during 4 minutes of 1 Hz dynamic muscle stretch and 4 minutes of static muscle stretch (performed in random order and separated by 10 minutes). Muscle stretch was performed by manually turning the rack and pinion. We found that triceps surae muscle interstitial PGE2 concentration was significantly increased during both static (↑38%, p=0.01) and dynamic (↑56%, p&lt;0.01) stretch above their respective baseline concentrations (n=9). Indomethacin (1μg/kg) significantly reduced baseline PGE2 concentration and prevented the increase in PGE2 during static (n=4) and dynamic (n=5) stretch. Importantly, indomethacin had no effect on the peak pressor response during static (control: 15±4, indomethacin: 16±4 mmHg; p=0.82) and dynamic (control: 17±2, indomethacin: 18±4 mmHg; p=0.68) stretch (n=7). Collectively, these data indicate that static and dynamic hindlimb muscle stretch in decerebrate, unanaesthetized rats increased muscle interstitial PGE2 concentration. The magnitude of increase in PGE2, however, was not sufficient to impact the muscle mechanoreflex.Support or Funding Information1R01HL142877 to SWCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic Femoral Artery Ligation Increases Skeletal Muscle Interstitial Prostaglandin E2 in the Rat: Implications for the Exercise Pressor Reflex

The stimulation of primarily mechanically‐sensitive group III and primarily metabolically‐sensitive group IV muscle afferents during skeletal muscle contraction evokes reflex increases in blood pressure (i.e., the exercise pressor reflex). The exercise pressor reflex is exaggerated in many forms of cardiovascular disease when compared to the reflex found in healthy counterparts. In rats in which a femoral artery is ligated for 72 hours (a model of simulated peripheral artery disease), the mechanically‐sensitive component of the reflex (i.e., the mechanoreflex) contributes importantly to the overall exercise pressor reflex exaggeration. Using a dynamic hindlimb muscle stretch protocol to study the mechanoreflex, we found recently that the increase in blood pressure was greater during stretch of a ligated versus a freely perfused hindlimb. This suggested that a chronic sensitization of mechanically‐activated channels contributed to the exaggerated mechanoreflex in ligated hindlimbs. The identity of the sensitizing chemical, however, was unknown. Experimental evidence has established that cyclooxygenase (COX) products, especially prostaglandins, sensitize mechanically‐activated channels. Accordingly, the purpose of this investigation was to determine whether 72 hour femoral artery ligation increased resting skeletal muscle interstitial prostaglandin E2 (PGE2) concentration in decerebrate, unanesthetized rats. The microdialysis technique was used to collect hindlimb skeletal muscle interstitial fluid in eight rats with a previously ligated femoral artery and a freely perfused femoral artery. We found that resting PGE2 concentration was greater in the ligated (224±44 pg/ml) versus the freely perfused (180±33 pg/ml, p&lt;0.01) hindlimb. To investigate the potential underlying mechanisms of the PGE2 elevation, we quantified both COX expression and COX activity in the soleus muscles (highly oxidative) and white portion of the gastrocnemius muscles (highly glycolytic). Western blot analyses revealed that femoral artery ligation had no effect on COX‐1 or COX‐2 protein expression in the soleus or white gastrocnemius muscles (p‐value range: 0.44 to 0.78). Total COX activity, however, was higher in the white gastrocnemius muscle of ligated hindlimbs compared to freely perfused hindlimbs (p&lt;0.04). There was no difference in total COX activity in the soleus muscle between hindlimbs (p=0.21). We conclude that 72 hours of femoral artery ligation increased resting PGE2 concentration in rat hindlimb skeletal muscles. The higher PGE2 concentration in ligated hindlimb muscles appears attributable to increased COX activity in white, glycolytic muscle portions. Our findings suggest that chronically elevated skeletal muscle interstitial PGE2 concentration may contribute to the exaggerated mechanically‐sensitive component of the exercise pressor reflex found in ligated hindlimb versus a freely perfused hindlimb.Support or Funding Information1R01HL142877 to SWCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Skeletal muscle interstitial fluid metabolomics at rest and associated with an exercise bout: application in rats and humans.

Blood or biopsies are often used to characterize metabolites that are modulated by exercising muscle. However, blood has inputs derived from multiple tissues, biopsies cannot discriminate between secreted and intracellular metabolites, and their invasive nature is challenging for frequent collections in sensitive populations (e.g., children and pregnant women). Thus, minimally invasive approaches to interstitial fluid (IF) metabolomics would be valuable. A catheter was designed to collect IF from the gastrocnemius muscle of acutely anesthetized adult male rats at rest or immediately following 20 min of exercise (~60% of maximal O2 uptake). Nontargeted, gas chromatography-time-of-flight mass spectrometry analysis was used to detect 299 metabolites, including nonannotated metabolites, sugars, fatty acids, amino acids, and purine metabolites and derivatives. Just 43% of all detected metabolites were common to IF and blood plasma, and only 20% of exercise-modified metabolites were shared in both pools, highlighting that the blood does not fully reflect the metabolic outcomes in muscle. Notable exercise patterns included increased IF amino acids (except leucine and isoleucine), increased α-ketoglutarate and citrate (which may reflect tricarboxylic acid cataplerosis or shifts in nonmitochondrial pathways), and higher concentration of the signaling lipid oleamide. A preliminary study of human muscle IF was conducted using a 20-kDa microdialysis catheter placed in the vastus lateralis of five healthy adults at rest and during exercise (65% of estimated maximal heart rate). Approximately 70% of commonly detected metabolites discriminating rest vs. exercise in rats were also changed in exercising humans. Interstitium metabolomics may aid in the identification of molecules that signal muscle work (e.g., exertion and fatigue) and muscle health.

Microdialysis Study of Aztreonam-Avibactam Distribution in Peritoneal Fluid and Muscle of Rats with or without Experimental Peritonitis.

The purpose of this study was to investigate aztreonam (ATM) and avibactam (AVI) distribution in intraperitoneal fluid and muscle interstitial fluid by microdialysis in rats, with or without peritonitis, and to compare the unbound concentrations in tissue with the unbound concentrations in blood. Microdialysis probes were inserted into the jugular veins, hind leg muscles, and peritoneal cavities of control rats (n = 5) and rats with intra-abdominal sepsis (n = 9) induced by cecal ligation and punctures. ATM and AVI probe recoveries in each medium were determined for both molecules in each rat by retrodialysis by drug. ATM-AVI combination was administered as an intravenous bolus at a dose of 100-25 mg · kg-1 Microdialysis samples were collected over 120 min, and ATM-AVI concentrations were determined by liquid chromatography-tandem mass spectrometry. Noncompartmental pharmacokinetic analysis was conducted and nonparametric tests were used for statistical comparisons between groups (infected versus control) and medium. ATM and AVI distribution in intraperitoneal fluid and muscle was rapid and complete both in control rats and in rats with peritonitis, and the concentration profiles in blood, intraperitoneal fluid, and muscle were virtually superimposed, in control and infected animals, both for ATM and AVI. No statistically significant difference was observed between unbound tissue extracellular fluid and systemic areas under the curve for both molecules in control and infected animals. In the present study, intraperitoneal infection induced by cecal ligation and puncture had no apparent effect on ATM and AVI pharmacokinetics in rats.

Design Considerations of a Fiber Optic Pressure Sensor Protective Housing for Intramuscular Pressure Measurements.

Intramuscular pressure (IMP), defined as skeletal muscle interstitial fluid pressure, reflects changes in individual muscle tension and may provide crucial insight into musculoskeletal biomechanics and pathologies. IMP may be measured using fiber-optic fluid pressure sensors, provided the sensor is adequately anchored to and shielded from surrounding muscle tissue. Ineffective anchoring enables sensor motion and inadequate shielding facilitates direct sensor-tissue interaction, which result in measurement artifacts and force-IMP dissociation. The purpose of this study was to compare the effectiveness of polyimide and nitinol protective housing designs to anchor pressure sensors to muscle tissue, prevent IMP measurement artifacts, and optimize the force-IMP correlation. Anchoring capacity was quantified as force required to dislodge sensors from muscle tissue. Force-IMP correlations and non-physiological measurement artifacts were quantified during isometric muscle activations of the rabbit tibialis anterior. Housing structural integrity was assessed after both anchoring and activation testing. Although there was no statistically significant difference in anchoring capacity, nitinol housings demonstrated greater structural integrity and superior force-IMP correlations. Further design improvements are needed to prevent tissue accumulation in the housing recess associated with artificially high IMP measurements. These findings emphasize fundamental protective housing design elements crucial for achieving reliable IMP measurements.

Effect of extraluminal ATP application on vascular tone and blood flow in skeletal muscle: implications for exercise hyperemia

During skeletal muscle contractions, the concentration of ATP increases in muscle interstitial fluid as measured by microdialysis probes. This increase is associated with the magnitude of blood flow, suggesting that interstitial ATP may be important for contraction-induced vasodilation. However, interstitial ATP has solely been described to induce vasoconstriction in skeletal muscle. To examine whether interstitial ATP induces vasodilation in skeletal muscle and to what extent this vasoactive effect is mediated by formation of nitric oxide (NO) and prostanoids, three different experimental models were studied. The rat gluteus maximus skeletal muscle model was used to study changes in local skeletal muscle hemodynamics. Superfused ATP at concentrations found during muscle contractions (1-10 μM) increased blood flow by up to 400%. In this model, the underlying mechanism was also examined by inhibition of NO and prostanoid formation. Inhibition of these systems abolished the vasodilator effect of ATP. Cell-culture experiments verified ATP-induced formation of NO and prostacyclin in rat skeletal muscle microvascular endothelial cells, and ATP-induced formation of NO in rat skeletal muscle cells. To confirm these findings in humans, ATP was infused into skeletal muscle interstitium of healthy subjects via microdialysis probes and found to increase muscle interstitial concentrations of NO and prostacyclin by ~60% and ~40%, respectively. Collectively, these data suggest that a physiologically relevant elevation in interstitial ATP concentrations increases muscle blood flow, indicating that the contraction-induced increase in skeletal muscle interstitial [ATP] is important for exercise hyperemia. The vasodilator effect of ATP application is mediated by NO and prostanoid formation.

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.

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.

The hyperaemic response to passive leg movement is dependent on nitric oxide: a new tool to evaluate endothelial nitric oxide function

Passive leg movement is associated with a ∼3-fold increase in blood flow to the leg but the underlying mechanisms remain unknown. The objective of the present study was to examine the role of nitric oxide (NO) for the hyperaemia observed during passive leg movement. Leg haemodynamics and metabolites of NO production (nitrite and nitrate; NOx) were measured in plasma and muscle interstitial fluid at rest and during passive leg movement with and without inhibition of NO formation in healthy young males. The hyperaemic response to passive leg movement and to ACh was also assessed in elderly subjects and patients with peripheral artery disease. Passive leg movement (60 r.p.m.) increased leg blood flow from 0.3 ± 0.1 to 0.9 ± 0.1 litre min(-1) at 20 s and 0.5 ± 0.1 litre min(-1) at 3 min (P < 0.05). Mean arterial pressure remained unchanged during the trial. When passive leg movement was performed during inhibition of NO formation (N(G)-mono-methyl-l-arginine; 29-52 mg min(-1)), leg blood flow and vascular conductance were increased after 20 s (P < 0.05) and then returned to baseline levels, despite an increase in arterial pressure (P < 0.05). Passive leg movement increased the femoral venous NOx levels from 35 ± 5 at baseline to 62 ± 11 μmol l(-1) during passive leg movement (P < 0.05), whereas muscle interstitial NOx levels remained unchanged. The hyperaemic response to passive leg movement were correlated with the vasodilatation induced by ACh (r(2) = 0.704, P < 0.001) and with age (r(2) = 0.612, P < 0.001). Leg blood flow did not increase during passive leg movement in individuals with peripheral arterial disease. These results suggest that the hypaeremia induced by passive leg movement is NO dependent and that the source of NO is likely to be the endothelium. Passive leg movement could therefore be used as a non-invasive tool to evaluate NO dependent endothelial function of the lower limb.

Pro‐ and anti‐angiogenic factors in human skeletal muscle in response to acute exercise and training

This study examined the effect of acute exercise and 4 weeks of aerobic training on skeletal muscle gene and protein expression of pro- and anti-angiogenic factors in 14 young male subjects. Training consisted of 60 min of cycling (∼60% of ), 3 times/week. Biopsies were obtained from vastus lateralis muscle before and after training. Muscle interstitial fluid was collected during cycling at weeks 0 and 4. Training increased (P < 0.05) the capillary: fibre ratio and capillary density by 23% and 12%, respectively. The concentration of interstitial vascular endothelial growth factor (VEGF) in response to acute exercise increased similarly (>6-fold; P < 0.05) before and after training. Resting protein levels of soluble VEGF receptor-1 in interstitial fluid, and of VEGF, thrombospondin-1 (TSP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP1) in muscle were unaffected by training, whereas endothelial nitric oxide synthase protein levels in muscle increased by 50% (P < 0.05). Before and after training, acute exercise induced a similar increase (P < 0.05) in the mRNA level of angiopoietin 2, matrix metalloproteinase 9 and TSP-1. After training, TIMP1 mRNA content increased with exercise (P < 0.05). In conclusion, acute exercise induced a similar increase in the gene-expression of both pro- and anti-angiogenic factors in untrained and trained muscle. We propose that the increase in anti-angiogenic factors with exercise is important for modulation of angiogenesis. The lack of effect of training on basal muscle VEGF protein levels and VEGF secretion during exercise suggests that increased VEGF levels are not a prerequisite for exercise-induced capillary growth in healthy muscle.

Is endothelial-independent vascular reactivity compromised in obese subjects with metabolic syndrome?

to the editor: Schinzari et al. ([4][1]) compared the endothelial-dependent and -independent vasodilatory responses in controls and subjects with metabolic syndrome (MetSyn). The primary objective was to explore the integrity of the nitric oxide (NO) pathway and evaluate the effects of

Muscular Angiogenic Factors and Capillary Growth - Changes with Intense Interval vs. Moderate Continuous Training.

PURPOSE: To examine the effect of a period of interval exercise training preceded by a period of continuous exercise training on angiogenic factors and capillarization in skeletal muscle. METHODS: Nine young males (intervention group) performed 60 min of continuous cycling at 62% of VO2 max 3 times/wk for 4 wks, followed by 4 wks of interval cycling consisting of 24 repetitions of 1 min bouts at 132% of VO2 max separated by 1.5 min of rest repeated 3 times/wk. In addition, five males performed 60 min of continuous cycling at 62% of VO2 max 3 times/wk for 8 wks (control group). Muscle biopsies were obtained from m. v. lateralis before as well as after 4 and 8 wks of training. Muscle interstitial fluid was collected during acute continuous cycling at week 0 and 4, and during acute interval cycling at week 4 and 8 in the intervention group. RESULTS: Intervention group: A bout of continuous exercise induced an increase in interstitial VEGF (106 ± 33 to 563 ± 131 pg/ml). A bout of interval cycling performed at 4 wks, resulted in a lower level of interstitial VEGF protein compared to continuous cycling (372 ± 107 and 690 ± 128 pg/ml, respectively; P < 0.01). Training had no effect on the magnitude of interstitial VEGF in response to exercise. Resting levels of muscle VEGF protein were 16 ± 4% lower (P < 0.05) after 4 and 8 wks of training compared to week 0 with no difference between week 4 and 8. The first 4 wks of training increased (50 ± 15%; P < 0.001) muscle eNOS protein levels and the levels were further elevated (30 ± 10%; P < 0.01) by the subsequent 4 wk period. Four wks of continuous training enhanced the capillary to fiber ratio (C:F) (2.37 ± 0.14 to 3.00 ± 0.26 cap fiber−2; P < 0.001) with no further change after 4 wks of interval training. Capillary density was elevated (433 ± 30 to 560 ± 40 cap mm−2; P < 0.05) after the first 4 wks and was further elevated (635 ± 35 cap mm−2; P < 0.05) after 4 wks of interval training. Control group: Muscle VEGF protein levels were not altered by training whereas eNOS protein levels were increased (P < 0.001) similarly as in the intervention group. C:F increased (2.67 ± 0.22 to 3.07 ± 0.21 cap fiber−2; P < 0.05) from 0 to 8 wks. CONCLUSION: The results demonstrate that the interstitial VEGF levels in response to exercise are dependent on the exercise mode but not on training status. A change in training mode from continuous moderate to intense interval training does not further enhance capillary growth or angiogenic factors compared to continued moderate exercise training.

The effect of passive movement training on angiogenic factors and capillary growth in human skeletal muscle

The effect of a period of passive movement training on angiogenic factors and capillarization in skeletal muscle was examined. Seven young males were subjected to passive training for 90 min, four times per week in a motor-driven knee extensor device that extended one knee passively at 80 cycles min₋₁. The other leg was used as control. Muscle biopsies were obtained from m. v. lateralis of both legs before as well as after 2 and 4 weeks of training. After the training period, passive movement and active exercise were performed with both legs, and muscle interstitial fluid was sampled from microdialysis probes in the thigh. After 2 weeks of training there was a 2-fold higher level of Ki-67 positive cells, co-localized with endothelial cells, in the passively trained leg which was paralleled by an increase in the number of capillaries around a fibre (P <0.05). Capillary density was higher than pre-training at 4 weeks of training (P <0.05). The training induced an increase in the mRNA level of endothelial nitric oxide synthase (eNOS), the angiopoietin receptor Tie-2 and matrix metalloproteinase (MMP)-9 in the passively trained leg and MMP-2 and tissue inhibitor of MMP (TIMP)-1 mRNA were elevated in both legs. Acute passive movement increased (P <0.05) muscle interstitial vascular endothelial growth factor (VEGF) levels 4- to 6-fold above rest and the proliferative effect, determined in vitro, of the muscle interstitial fluid ~16-fold compared to perfusate. The magnitude of increase was similar for active exercise. The results demonstrate that a period of passive movement promotes endothelial cell proliferation and angiogenic factors and initiates capillarization in skeletal muscle.

Effect of Osteopathic Manipulation on Interstitial Concentrations of Inflammatory Markers

Little is known about the physiology underlying the benefits of osteopathic manipulative treatment (OMT). In vitro studies demonstrate a potential anti-inflammatory effect of OMT. PURPOSE: This research investigated the effects of OMT on inflammatory markers in rat skeletal muscle interstitial fluid, in vivo. METHODS: Inflammation was stimulated via bilateral, intra-articular injection of sodium iodoacetate into the knee joints of adult Sprague Dawley rats (n=8) to induce osteoarthritis. Animals were randomized to OMT or sham treatment four weeks post-injection. OMT included modified muscle energy treatments consisting of two techniques: 1) a practitioner restrained the rat's body and forelimbs. The rat extended its hindlimbs in an attempted escape maneuver, during which the hindlimb pushed against the practitioner's finger; and 2) the hindlimb was pulled into maximal extension. Light pressure applied to the foot evoked a reflexive flexion, against which the practitioner provided slight resistance. Passive myofascial stretching was conducted by applying light tensile force to the extended hindlimb. This force was held until a release was detected. Indirect techniques were held for 20 seconds, and direct techniques were repeated 3 times. Microdialysis probes were placed in the biceps femoris to collect cytokines from the muscular interstitium for 2 hours during each condition (pre-arthritis, post-arthritis, and post-OMT). Interstitial cytokine concentrations were determined using Luminex xMAP technology. RESULTS: Compared with baseline interstitial cytokine concentrations, IL-1 beta was significantly higher in the arthritis condition (p < 0.05). IL-6 and IL-13 tended to be higher in the arthritic condition, but did not reach statistical significance (IL-6, p=0.066, IL-13, p=0.08). However, in the subset of four animals that received OMT, IL-13 was significantly higher in both the arthritis conditions and post-OMT conditions, compared with baseline (p < 0.05). CONCLUSIONS: These preliminary findings suggest that certain cytokines measured in skeletal muscle interstitial fluid are elevated in response to induction of osteoarthritis, and that at least one cytokine, IL-13, continues to increase in response to OMT, suggesting a potential pathway by which OMT may elicit its purported effects.

Cytokines are produced locally by myocytes in rat skeletal muscle during endotoxemia

Cytokines act as chemical mediators during the inflammatory process. Measurements of cytokine levels in tissue have previously been performed in homogenized tissue, but the true concentrations in native interstitial fluid (ISF), i.e., the compartment where cytokines exert their biologically active role, have remained unknown. The role of skeletal muscle myocytes as a source for cytokines during endotoxemia was explored by collecting muscle ISF using a wick method, and the levels of 14 cytokines in ISF and plasma were related to the corresponding changes in mRNA levels to reveal any potential discrepancies between gene expression and protein release of cytokines to ISF. The majority of investigated cytokines were elevated in muscle ISF during endotoxemia, and an analysis of cytokine mRNA levels revealed consistency between gene expression and protein release. The elevated cytokine level in ISF, in addition to elevated gene expression in muscle, indicated a significant local production and release of several proinflammatory cytokines and chemokines within skeletal muscle tissue during endotoxemia. Immunohistochemistry revealed that myocytes constituted a significant source of IL-1beta and TNF-alpha production during endotoxemia, whereas the contribution from inflammatory cells i.e., leukocytes, was found to be less significant. Muscle cells apparently constitute an important source of several different cytokines during endotoxemia, governing the level in the muscle microenvironment, and are likely to contribute significantly to cytokine levels in plasma.