Skeletal Muscle Capillary Density Research Articles

Endothelial-Specific Reduction in Arf6 Impairs Insulin-Stimulated Vasodilation and Skeletal Muscle Blood Flow Resulting in Systemic Insulin Resistance in Mice.

Much of what we know about insulin resistance is based on studies from metabolically active tissues such as the liver, adipose tissue, and skeletal muscle. Emerging evidence suggests that the vascular endothelium plays a crucial role in systemic insulin resistance; however, the underlying mechanisms remain incompletely understood. Arf6 (ADP ribosylation factor 6) is a small GTPase that plays a critical role in endothelial cell function. Here, we tested the hypothesis that the deletion of endothelial Arf6 will result in systemic insulin resistance. We used mouse models of constitutive endothelial cell-specific Arf6 deletion (Arf6f/- Tie2Cre+) and tamoxifen-inducible Arf6 knockout (Arf6f/f Cdh5CreER+). Endothelium-dependent vasodilation was assessed using pressure myography. Metabolic function was assessed using a battery of metabolic assessments including glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps. We used a fluorescence microsphere-based technique to measure tissue blood flow. Skeletal muscle capillary density was assessed using intravital microscopy. Endothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide bioavailability but independent of altered acetylcholine-mediated or sodium nitroprusside-mediated vasodilation. Endothelial cell-specific deletion of Arf6 also resulted in systematic insulin resistance in normal chow-fed mice and glucose intolerance in high-fat diet-fed obese mice. The underlying mechanisms of glucose intolerance were reductions in insulin-stimulated blood flow and glucose uptake in the skeletal muscle and were independent of changes in capillary density or vascular permeability. Results from this study support the conclusion that endothelial Arf6 signaling is essential for maintaining insulin sensitivity. Reduced expression of endothelial Arf6 impairs insulin-mediated vasodilation and results in systemic insulin resistance. These results have therapeutic implications for diseases that are associated with endothelial cell dysfunction and insulin resistance such as diabetes.

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.

Abstract 15747: AMPK Cysteine Oxidation by SOD3-Derived H 2 O 2 Mediates Exercise-Induced Restoration of Skeletal Muscle Angiogenesis in Type2 Diabetes

Background: Exercise restores decreased capillary density in skeletal muscle (SKM) (capillary rarefaction) in type2 diabetes (T2D) that has eNOS uncoupling/excess O 2 - , which is required for treatment of diabetic peripheral vascular disease (PAD) via unknown mechanisms. AMP-activated protein kinase (AMPK) is a key angiogenic/metabolic kinase mainly regulated by phosphorylation and is required for baseline, but not exercise-induced capillary density in SKM under healthy conditions. AMPK senses H 2 O 2 signal to increase its activity via oxidative modification of Cys299/304 in vitro. In addition, extracellular SOD (SOD3) that catalyzes the dismutation of O 2 - to H 2 O 2 is increased in response to exercise. However, the role of AMPK and SOD3 in exercise-induced SKM angiogenesis in T2D has not been reported. Oxidation of Cysteine residues to generate cysteine sulfenic acid (Cys-OH) is a key event in H 2 O 2 -mediated signaling. Results: Here we show that reduced CD31+ capillary density in SKM of high fat diet-induced T2D mice was restored (2.4-fold) by voluntary wheel exercise training (2 weeks) with enhanced SOD3 activity (224%) and extracellular H 2 O 2 levels (3.2-fold). Of note, exercise in T2D mice increased AMPK activity (by p-ACC) via inducing CysOH formation of AMPK (3.6-fold), but not p-AMPK induction, which was inhibited by SOD3 -/- /T2D mice that reduced exercise-induced H 2 O 2 . To demonstrate functional significance of AMPK Cys oxidation, we generated CRISPR/Cas9-genome editing “redox-dead” AMPK-Cys299/304Ala (AMPK-C/A) knock-in (KI) mutant mice and found that AMPK-C/A KI mice showed significant inhibition of exercise-induced angiogenesis (44%) via reducing angiogenic PGC1α and VEGF protein expression in SKM of T2D mice. In vitro, C2C12 skeletal muscles overexpressing SOD3 treated with palmitate (mimic T2D with exercise) induced AMPK-CysOH formation and VEGF secretion. Its conditioned media promoted angiogenic responses such as EC migration (1.8-fold) and tube formation (1.7-fold), which was inhibited by C2C12 cells overexpressing AMPK C/A mutant. Conclusion: Novel exercise-induced AMPK CysOH formation at Cys299/304 restores angiogenesis in SKM of T2D via SOD3-mediated H 2 O 2 , which may contribute to revascularization in diabetic PAD.

1372-P: Hyperglycemia Impacts Mouse Skeletal Muscle in a Sex-Specific Manner

Functional Exercise Capacity (FEC) , a universal predictor of cardiovascular and all-cause mortality, is lower in adults and youth with diabetes for reasons that are poorly understood. Skeletal muscle is key determinant of FEC. We hypothesized that skeletal muscle capillary and mitochondrial density are lower with hyperglycemia and respond to exercise training. To test this hypothesis we generated a model of short-term hyperglycemia (HG) with inducible beta cell deletion of glucokinase (GKlox/lox;PdxCreER) . Male (M) and female (F) HG mice and euglycemic littermate controls (EG) completed 3 weeks of treadmill training (Ex) or remained sedentary (Sed) . The gastrocnemius was collected 24 hours after the final exercise bout. Expression of COX1 (mitochondrial marker) , Dystrophin (muscle fiber sarcolemma boundary marker) , and CD31 (capillary marker) was detected by immunofluorescence. Sirtuin 3 (SIRT3) , an NAD+ dependent deacetylase (mitochondrial homeostasis) was measured using the JESS system. (Fig 1) Mitochondrial density (mitochondria per fiber as quantified by fluorescence intensity units per fiber [FIU/fiber]) was significantly lower in HG vs. EG (p<0.0001) and differed by sex (p=0.0048 sex x glycemia interaction) . Ex resulted in greater mitochondrial density (p=0.0265) independent of sex and glycemia. Capillary density and SIRT3 were unchanged. Sex-differences in muscle response to HG warrant further investigation. Disclosure L.Knaub: None. G.Pott: None. N.A.Hulett: None. S.Hull: None. R.L.Scalzo: None. J.Reusch: Advisory Panel; Medtronic. Funding VA Merit (BX002046)

Tetrahydrobiopterin synthesis and metabolism is impaired in dystrophin-deficient mdx mice and humans.

Loss of dystrophin causes oxidative stress and affects nitric oxide synthase-mediated vascular function in striated muscle. Because tetrahydrobiopterin is an antioxidant and co-factor for nitric oxide synthase, we tested the hypothesis that tetrahydrobiopterin would be low in mdx mice and humans deficient for dystrophin. Tetrahydrobiopterin and its metabolites were measured at rest and in response to exercise in Duchenne and Becker muscular dystrophy patients, age-matched male controls as well as wild-type, mdx and mdx mice transgenically overexpressing skeletal muscle-specific dystrophins. Mdx mice were also supplemented with tetrahydrobiopterin and pathophysiology was assessed. Duchenne muscular dystrophy patients had lower urinary dihydrobiopterin+tetrahydrobiopterin/specific gravity1.020 compared to unaffected age-matched males and Becker muscular dystrophy patients. Mdx mice had low urinary and skeletal muscle dihydrobiopterin+tetrahydrobiopterin compared to wild-type mice. Overexpression of dystrophins that localize neuronal nitric oxide synthase restored dihydrobiopterin+tetrahydrobiopterin in mdx mice to wild-type levels while utrophin overexpression did not. Mdx mice and Duchenne muscular dystrophy patients did not increase tetrahydrobiopterin during exercise and in mdx mice tetrahydrobiopterin deficiency was likely because of lower levels of sepiapterin reductase in skeletal muscle. Tetrahydrobiopterin supplementation improved skeletal muscle strength, resistance to fatiguing and injurious contractions in vivo, increased utrophin and capillary density of skeletal muscle and lowered cardiac muscle fibrosis and left ventricular wall thickness in mdx mice. These data demonstrate that impaired tetrahydrobiopterin synthesis is associated with dystrophin loss and treatment with tetrahydrobiopterin improves striated muscle histopathology and skeletal muscle function in mdx mice.

Skeletal muscle capillary density is related to anaerobic threshold and claudication in peripheral artery disease.

Peripheral artery disease (PAD) is characterized by impaired blood flow to the lower extremities, causing claudication and exercise intolerance. Exercise intolerance may result from reduced skeletal muscle capillary density and impaired muscle oxygen delivery. This cross-sectional study tested the hypothesis that capillary density is related to claudication times and anaerobic threshold (AT) in patients with PAD. A total of 37 patients with PAD and 29 control subjects performed cardiopulmonary exercise testing on a treadmill for AT and gastrocnemius muscle biopsies. Skeletal muscle capillary density was measured using immunofluorescence staining. PAD had decreased capillary density (278 ± 87 vs 331 ± 86 endothelial cells/mm2, p = 0.05), peak VO2 (15.7 ± 3.9 vs 24.3 ± 5.2 mL/kg/min, p ⩽ 0.001), and VO2 at AT (11.5 ± 2.6 vs 16.1 ± 2.8 mL/kg/min, p ⩽ 0.001) compared to control subjects. In patients with PAD, but not control subjects, capillary density was related to VO2 at AT (r = 0.343; p = 0.038), time to AT (r = 0.381; p = 0.020), and time after AT to test termination (r = 0.610; p ⩽ 0.001). Capillary density was also related to time to claudication (r = 0.332; p = 0.038) and time after claudication to test termination (r = 0.584; p ⩽ 0.001). In conclusion, relationships between capillary density, AT, and claudication symptoms indicate that, in PAD, exercise limitations are likely partially dependent on limited skeletal muscle capillary density and oxidative metabolism.

The comparing effects of four-week rock climbing with or without blood flow restriction on vascular endothelial growth factor and Growth Hormone in elite climbers

Background: Angiogenesis and increased capillary density of skeletal muscle is one of the potential physiological changes during the flow restriction exercise (BFR). Therefore, the aim of this research was to evaluate the effect of four weeks rock climbing with or without blood flow restriction on vascular endothelial growth factor and Growth Hormone in elite climbers. Methods: In this semi-experimental study, 26 elite climbers (aged 25-30 years; body fat percent 8-14%; 4 years athletic training history) in a randomized and double-blind design were divided in two equal rock climbing with blood flow restriction (BFR) or rock climbing without blood flow restriction (NBFR) groups. The training protocol included three sessions per week for 4 weeks of rock climbing with a severity of 80-60% of the difficulty of the route. The cuff pressure was in the range of 40 to 100 mm Hg during rock climbing. Blood samples were obtained in the 2 phases: before and 24 hours after rock climbing protocols. Vascular endothelial growth factor and Growth Hormone were analyzed. Finally, Data were analyzed independent and independent T test. The significance level was set at p<0.05. Results: Basal levels of GH and VEGF was significantly increased after four weeks rock climbing with BFR. However, the rock climbing without BFR had not any significant effect on the basal levels of GH and VEGF. Conclusion: It can be concluded that the rock climbing with BFR by increasing of basal levels of GH and VEGF could be increased angiogenesis process.

Attenuation of Mineralocorticoid Receptor Signaling Promotes Vascular Recovery from Hind Limb Ischemia in a Sex Specific Manner

Peripheral arterial disease (PAD) is a growing concern affecting 8.5 million Americans annually and contributing to both fatal and non‐fatal cardiovascular ischemic events. Importantly, available evidence indicates that women experience higher rates of occlusion, reintervention, and mortality than males, however, mechanisms underlying these sex differences remain unclear. Recent evidence has revealed sex‐specific deleterious effects of mineralocorticoid receptor (MR) signaling in a variety of cardiovascular conditions including vascular inflammation and endothelial dysfunction, which have been implicated in PAD. Whether MR signaling differentially impairs microvascular adaptations in males and females following an ischemic insult, however, has not been examined. Accordingly, we evaluated the hypothesis that attenuation of MR signaling promotes microvascular compensation and perfusion recovery following hind limb ischemia (HLI) to a greater extent in females than in males involving enhanced capillary neovascularization and augmented outward remodeling of existing collaterals. Male and female C57BL/6J mice (10–12 weeks old) underwent unilateral femoral artery ligation and excision to induce HLI after implantation of pellets containing either placebo or the MR antagonist spironolactone (20mg/kg/day, sc). The contralateral leg underwent sham surgery with no ligation or excision. Hind limb perfusion was serially assessed for 28 days with laser Doppler imaging. At 28 days post occlusion, the hind limb vasculature was perfusion fixed following maximal dilation and perfused with bismuth for assessment of collateral morphology by micro‐CT imaging. These studies were complemented with immunohistochemistry and angiogenic assay experiments. Additional preliminary HLI experiments were conducted in male mice with endothelial cell‐specific MR (EC MR) deletion. Systemic inhibition of MR signaling enhanced recovery of hind limb perfusion in female, not male, mice. This effect of MR blockade was associated with greater outward collateral remodeling in females compared to males. Interestingly, spironolactone similarly increased skeletal muscle capillary density in both hind limbs of males and females consistent with impairment of VEGF‐stimulated capillary sprouting by the MR agonist aldosterone in an aortic angiogenesis assay. Finally, preliminary studies revealed enhanced recovery of hind limb perfusion in male mice with EC MR deletion and that EC MR deletion enhanced aortic capillary sprouting in vitro. Taken together, these data suggest that MR signaling limits the microvascular response to ischemia and that enhanced perfusion recovery following MR blockade in females involves greater collateral expansion compared to males. Significant vascular cell‐specificity of MR signaling underlying these responses may contribute to these sex differences and warrant further investigation.Support or Funding InformationFunded by HL136386, HL095590, and HL088105.

Lifelong Endurance Exercise as a Countermeasure Against Age-Related [Formula: see text] Decline: Physiological Overview andInsights from Masters Athletes.

Maximum oxygen consumption ([Formula: see text]) is not only an indicator of endurance performance, but also a strong predictor of cardiovascular disease and mortality. This physiological parameter is known to decrease with aging. In turn, physical exercise might attenuate the rate of aging-related decline in [Formula: see text], which in light of the global population aging is of major clinical relevance, especially at advanced ages. In this narrative review, we summarize the evidence available from masters athletes about the role of lifelong endurance exercise on aging-related [Formula: see text] decline, with examples of the highest [Formula: see text] values reported in the scientific literature for athletes across different ages (e.g., 35ml·kg-1·min-1 in a centenarian cyclist). These data suggest that a linear decrease in [Formula: see text] might be possible if physical exercise loads are kept consistently high through the entire life span, with [Formula: see text] values remaining higher than those of the general population across all ages. We also summarize the main physiological changes that occur with inactive aging at different system levels-pulmonary and cardiovascular function, blood O2 carrying capacity, skeletal muscle capillary density and oxidative capacity-and negatively influence [Formula: see text], and review how lifelong exercise can attenuate or even prevent most-but apparently not all (e.g., maximum heart rate decline)-of them. In summary, although aging seems to be invariably associated with a progressive decline in [Formula: see text], maintaining high levels of physical exercise along the life span slows the multi-systemic deterioration that is commonly observed in inactive individuals, thereby attenuating age-related [Formula: see text] decline.

Human Galectin-1 Improves Sarcolemma Stability and Muscle Vascularization in the mdx Mouse Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a devastating disease caused by mutations in the dystrophin gene that result in the complete absence of dystrophin protein. We have shown previously that recombinant mouse Galectin-1 treatment improves physiological and histological outcome measures in the mdx mouse model of DMD. Because recombinant human Galectin-1 (rHsGal1) will be used to treat DMD patients, we performed a dose-ranging study and intraperitoneal or intravenous delivery to determine the efficacy of rHsGal1 to improve preclinical outcome measures in mdx mice. Our studies showed that the optimal dose of rHsGal1 delivered intraperitoneally was 20 mg/kg and that this treatment improved muscle strength, sarcolemma stability, and capillary density in skeletal muscle. We next examined the efficacy of intravenous delivery and found that a dose of 2.5 mg/kg rHsGal1 was well tolerated and improved outcome measures in the mdx mouse model. Our studies identified that intravenous doses of rHsGal1 exceeding 2.5 mg/kg resulted in toxicity, indicating that dosing using this delivery mechanism will need to be carefully monitored. Our results support the idea that rHsGal1 treatment can improve outcome measures in the mdx mouse model and support further development as a potential therapeutic agent for DMD.

Increased capillary density in skeletal muscle is not associated with impaired insulin sensitivity induced by bed rest in healthy young men

Physical inactivity alters glucose homeostasis in skeletal muscle, potentially developing into overt metabolic disease. The present study sought to investigate the role of skeletal muscle capillarization in glucose tolerance and insulin sensitivity (IS) using a classic human model of physical inactivity. Thirteen healthy males (age = 23 ± 2 years) underwent 4 days of full-time supervised and diet-controlled bed rest. Oral glucose tolerance test, indices of IS (quantitative insulin sensitivity check index (QUICKI), Matsuda index), as well as skeletal muscle biopsies with measurement of fiber type distribution, fiber cross-sectional area (FCSA), capillary-to-fiber ratio (C/F ratio), and capillary density (CD) were assessed prior to and after bed rest. Body weight and composition were unaltered by bed rest. Fasting glucose/insulin ratio (G0/I0 ratio) (-25%, P = 0.016), QUICKI (-7%, P = 0.023), and Matsuda index (-24%, P = 0.003) diminished with bed rest. Skeletal muscle FCSA decreased (-737.4 ± 763.2 μm-2 (-12%), P = 0.005) while C/F ratio was preserved, resulting in augmented CD (+93.9 ± 91.5 capillaries·mm-2 (+37%), P = 0.003) with bed rest. No association was detected between changes in skeletal muscle variables and metabolic outcomes. Independently of bed rest-induced effects, a positive linear relationship was detected between C/F ratio and G0/I0 ratio (β = 17.09, P = 0.021). In conclusion, impaired glucose homeostasis with bed rest is not prevented nor associated with enhanced skeletal muscle capillarization in healthy individuals.

Akap1 Regulates Vascular Function and Endothelial Cells Behavior.

MitoAKAPs (mitochondrial A kinase anchoring proteins), encoded by the Akap1 gene, regulate multiple cellular processes governing mitochondrial homeostasis and cell viability. Although mitochondrial alterations have been associated to endothelial dysfunction, the role of mitoAKAPs in the vasculature is currently unknown. To test this, postischemic neovascularization, vascular function, and arterial blood pressure were analyzed in Akap1 knockout mice (Akap1-/- ) and their wild-type (wt) littermates. Primary cultures of aortic endothelial cells (ECs) were also obtained from Akap1-/- and wt mice, and ECs migration, proliferation, survival, and capillary-like network formation were analyzed under different experimental conditions. After femoral artery ligation, Akap1-/- mice displayed impaired blood flow and functional recovery, reduced skeletal muscle capillary density, and Akt phosphorylation compared with wt mice. In Akap1-/- ECs, a significant enhancement of hypoxia-induced mitophagy, mitochondrial dysfunction, reactive oxygen species production, and apoptosis were observed. Consistently, capillary-like network formation, migration, proliferation, and AKT phosphorylation were reduced in Akap1-/- ECs. Alterations in Akap1-/- ECs behavior were also confirmed in Akap1-/- mice, which exhibited a selective reduction in acetylcholine-induced vasorelaxation in mesenteric arteries and a mild but significant increase in arterial blood pressure levels compared with wt. Finally, overexpression of a constitutively active Akt mutant restored vascular reactivity and ECs function in Akap1-/- conditions. These results demonstrate the important role of mitoAKAPs in the modulation of multiple ECs functions in vivo and in vitro, suggesting that mitochondria-dependent regulation of ECs might represent a novel therapeutic approach in cardiovascular diseases characterized by endothelial dysfunction.

Exercise training dose differentially alters muscle and heart capillary density and metabolic functions in an obese rat with metabolic syndrome.

What is the central question of this study? Regular exercise is recommended as a non-pharmacological approach for the prevention and treatment of metabolic syndrome. However, the impact of different combinations of intensity, duration and frequency of exercise on metabolic syndrome and microvascular density has not been reported. What is the main finding and its importance? We provide evidence on the impact of aerobic exercise dose on metabolic and microvascular alterations in an experimental model of metabolic syndrome induced by high-fat diet. We found that the exercise frequency and duration were the main factors affecting anthropometric and metabolic parameters and microvascular density in the skeletal muscle. Exercise intensity was related only to microvascular density in the heart. We evaluated the effect of the frequency, duration and intensity of exercise training on metabolic parameters and structural capillary density in obese rats with metabolic syndrome. Wistar-Kyoto rats were fed either a standard commercial diet (CON) or a high-fat diet (HFD). Animals that received the HFD were randomly separated into either a sedentary (SED) group or eight different exercise groups that varied according to the frequency, duration and intensity of training. After 12weeks of aerobic exercise training, the body composition, aerobic capacity, haemodynamic variables, metabolic parameters and capillary density in the heart and skeletal muscle were evaluated. All the exercise training groups showed reduced resting systolic blood pressure and heart rate and normalized fasting glucose. The minimal amount of exercise (90minper week) produced little effect on metabolic syndrome parameters. A moderate amount of exercise (150minper week) was required to reduce body weight and improve capillary density. However, only the high amount of exercise (300minper week) significantly reduced the amount of body fat depots. The three-way ANOVA showed a main effect of exercise frequency and duration for the improvement of metabolic syndrome and capillary density in skeletal muscle. Exercise intensity was a main factor in reversing microvascular rarefaction in the heart.

Reduced fetal vitamin D status by maternal undernutrition during discrete gestational windows in sheep.

Placental transport of vitamin D and other nutrients (e.g. amino acids, fats and glucose) to the fetus is sensitive to maternal and fetal nutritional cues. We studied the effect of maternal calorific restriction on fetal vitamin D status and the placental expression of genes for nutrient transport [aromatic T-type amino acid transporter-1 (TAT-1); triglyceride hydrolase/lipoprotein uptake facilitator lipoprotein lipase (LPL)] and vitamin D homeostasis [CYP27B1; vitamin D receptor (VDR)], and their association with markers of fetal cardiovascular function and skeletal muscle growth. Pregnant sheep received 100% total metabolizable energy (ME) requirements (control), 40% total ME requirements peri-implantation [PI40, 1-31 days of gestation (dGA)] or 50% total ME requirements in late gestation (L, 104-127 dGA). Fetal, but not maternal, plasma 25-hydroxy-vitamin D (25OHD) concentration was lower in PI40 and L maternal undernutrition groups (P<0.01) compared with the control group at 0.86 gestation. PI40 group placental CYP27B1 messenger RNA (mRNA) levels were increased (P<0.05) compared with the control group. Across all groups, higher fetal plasma 25OHD concentration was associated with higher skeletal muscle myofibre and capillary density (P<0.05). In the placenta, higher VDR mRNA levels were associated with higher TAT-1 (P<0.05) and LPL (P<0.01) mRNA levels. In the PI40 maternal undernutrition group only, reduced fetal plasma 25OHD concentration may be mediated in part by altered placental CYP27B1. The association between placental mRNA levels of VDR and nutrient transport genes suggests a way in which the placenta may integrate nutritional cues in the face of maternal dietary challenges and alter fetal physiology.

Physiological adaptations to interval training and the role of exercise intensity.

Interval exercise typically involves repeated bouts of relatively intense exercise interspersed by short periods of recovery. A common classification scheme subdivides this method into high-intensity interval training (HIIT; 'near maximal' efforts) and sprint interval training (SIT; 'supramaximal' efforts). Both forms of interval training induce the classic physiological adaptations characteristic of moderate-intensity continuous training (MICT) such as increased aerobic capacity (V̇O2 max ) and mitochondrial content. This brief review considers the role of exercise intensity in mediating physiological adaptations to training, with a focus on the capacity for aerobic energy metabolism. With respect to skeletal muscle adaptations, cellular stress and the resultant metabolic signals for mitochondrial biogenesis depend largely on exercise intensity, with limited work suggesting that increases in mitochondrial content are superior after HIIT compared to MICT, at least when matched-work comparisons are made within the same individual. It is well established that SIT increases mitochondrial content to a similar extent to MICT despite a reduced exercise volume. At the whole-body level, V̇O2 max is generally increased more by HIIT than MICT for a given training volume, whereas SIT and MICT similarly improve V̇O2 max despite differences in training volume. There is less evidence available regarding the role of exercise intensity in mediating changes in skeletal muscle capillary density, maximum stroke volume and cardiac output, and blood volume. Furthermore, the interactions between intensity and duration and frequency have not been thoroughly explored. While interval training is clearly a potent stimulus for physiological remodelling in humans, the integrative response to this type of exercise warrants further attention, especially in comparison to traditional endurance training.

Angiogenesis and Expression of IGF2R, IGF2, uPAR, and TGFB in a Rat Obesity Model

IntroductionOur group has previously shown that obesity decreases capillary density in skeletal muscle. Here, we tested the hypothesis that decreased capillary density in obesity is the result of increased activity of the insulin growth factor II receptor (IGF2R) in degrading urokinase‐plasminogen receptor (uPAR), activating transforming growth factor beta (TGFB), and degrading insulin growth factor II (IGF2) using an obese, diabetic rat model.MethodsAt four weeks of age, normophagic Long‐Evans Tokyushima Otsuka (LETO; n=20) and hyperphagic Otsuka Long‐Evans Tokushima Fatty (OLETF; n=20) rats were provided with either a normal, low‐fat diet (LFD, 10% kcal fat) or a high‐fat, high‐cholesterol diet (HFD, 45%kcal fat with 1% cholesterol). At 32 weeks of age, samples from rats were taken, including biceps brachii and vastus lateralis muscle, and arterial vessels. Whole tissue muscle samples were snapfrozen and cold sectioned. Slides were stained for IHC highlighting of capillaries, with manual counting of capillaries and muscle fibers in cross‐section. Vessels were homogenized in buffer and processed by way of Western blot procedure, with membranes probed for IGF2R, IGF2, TGFB, and uPAR.ResultsCapillary density in biceps brachii was reduced in OLETF LFD rats (p&lt;0.07) compared to LETO LFD rats, but HFD increased capillary density significantly relative to LFD counterpart, especially in OLETF rats (19%, p&lt;0.01). In addition, vastus lateralis IHC indicated a significant decrease in capillary density in OLETF LFD/HFD and LETO HFD relative to LETO LFD (p&lt;0.05). Western blot analysis of arterial vessel samples did not indicate any significant difference in IGF2R, IGF2, TGFB, or uPAR between LETO LFD and other groups.DiscussionThese data demonstrate that capillary density is significantly downregulated in obese, type 2 diabetic OLETF rats. However, examination of arterial samples suggest that changes may not be related to changes in expression of IGF2R and associated proposed down‐regulators of vascular growth in the arterial vasculature. To complete the test of our hypothesis we will measure IGF2R, uPAR, IGF2, and TGFB expression in muscle tissue to determine whether changes in expression occur in the muscle that may mediate changes in capillarity in obesity/diabetes.Support or Funding InformationWe Acknowledge Support of this research: NIH HL36088(MHL), VHA CDA2 1BX001299 (RSR), Society of Phi Zeta, and the University of Missouri Department of Medical Pharmacology and Physiology.

Advances and challenges in skeletal muscle angiogenesis.

The role of capillaries is to serve as the interface for delivery of oxygen and removal of metabolites to/from tissues. During the past decade there has been a proliferation of studies that have advanced our understanding of angiogenesis, demonstrating that tissue capillary supply is under strict control during health but poorly controlled in disease, resulting in either excessive capillary growth (pathological angiogenesis) or losses in capillarity (rarefaction). Given that skeletal muscle comprises nearly 40% of body mass in humans, skeletal muscle capillary density has a significant impact on metabolism, endocrine function, and locomotion and is tightly regulated at many different levels. Skeletal muscle is also high adaptable and thus one of the few organ systems that can be experimentally manipulated (e.g., by exercise) to study physiological regulation of angiogenesis. This review will focus on the methodological concerns that have arisen in determining skeletal muscle capillarity and highlight the concepts that are reshaping our understanding of the angio-adaptation process. We also summarize selected new findings (physical influences, molecular changes, and ultrastructural rearrangement of capillaries) that identify areas of future research with the greatest potential to expand our understanding of how angiogenesis is normally regulated, and that may also help to better understand conditions of uncontrolled (pathological) angiogenesis.

Reduced uterine perfusion pressure decreases functional capillary density in skeletal muscle.

The purpose of this study was to examine the functional and structural capillary density in the reduced uterine perfusion pressure (RUPP) model, which when performed during pregnancy is an established animal model of preeclampsia. We hypothesized that the RUPP model would be associated with capillary rarefaction and impaired capillary perfusion, which would be more pronounced in the pregnant state. Female Sprague-Dawley rats (n = 32) were randomized to nonpregnancy (Nonpregnant) or breeding (Pregnant) at 12 wk of age and again to RUPP or SHAM surgeries on gestational day (GD) 14 (or equivalent age in nonpregnant rats). On GD 20 (or equivalent), capillary structure and perfusion of the extensor digitorum longus were imaged using digital intravital video microscopy. Functional videos were analyzed by a blinded observer to measure capillary density, expressed as capillaries per millimeter intersecting three staggered reference lines (200 μm). Flow was scored as the percentage of capillaries having 1) continuous, 2) intermittent, or 3) stopped flow. Total capillary density was not different between groups. There was a main effect of RUPP surgery resulting in decreased continuous flow vessels (P < 0.01) and increased stopped flow (P < 0.01), which was driven by differences between pregnant animals (Continuous flow: pregnant SHAM 80.1 ± 7.8% vs. pregnant RUPP 67.8 ± 11.2%, P < 0.05) (Stopped flow: pregnant SHAM 8.7 ± 3.2% vs. pregnant RUPP 17.9 ± 5.7%, P < 0.01). Our results demonstrate that the RUPP surgery is associated with a decrease in functional capillary density in skeletal muscle that is more pronounced in the pregnant state, which may contribute to the vascular pathophysiology observed in preeclampsia.

Abstract 10: MicroRNA 17 in Angiogenesis: Lessons Learned From Immobilized Vascular Endothelial Growth Factor

Several pre-clinical and clinical studies are exploring the therapeutic effect of cell-based therapies in ischemic diseases, including Peripheral Artery Disease (PAD). Unfortunately, most of the cells (more than 80%) die few days after delivery. We postulate that better understanding of VEGF Biology might be important for the design of more effective pro-survival strategies. Release of VEGF from ECM activates a carcinogenic program since it triggers vasculogenesis, tumor growth and metastasis. Contrarily, immobilized VEGF might have all the properties of soluble VEGF without inducing a carcinogenic program. Thus, identification of downstream players such as miRNAs mediating this process might be important. Herein, we evaluated therapeutic effect of miR-17 downregulation, which mediates the effect of immobilized VEGF both in vitro and in vivo. We recently showed that conjugated VEGF modulates cell activity by decreasing the expression miR-17 both in vitro and in vivo. In the present study, cell survival and angiogenesis were evaluated firstly in vitro using endothelial cells (ECs) transfected with antagomiR-17 to mimic the down-regulation of miR-17 by conjugated VEGF. AntagomiR-17 increased EC survival at least 1.5 times (n=6) compared to pro-angiogenic miRNAs reported in the literature (e.g. miR-424 and miR-132) and sprout formation on Matrigel at least 2 times (n=5) compared to all groups. The effect of antagomiR-17 was more pronounced under hypoxia conditions. In vivo, antagomiR-17 accelerated hemodynamic recovery of the whole limb (n=12) in unilateral limb ischemia obtained by occlusion of the left femoral artery. Blood flow recovery evaluated by Laser Doppler analysis was significantly higher 21 days after surgery in antagomiR-17 group compared to all other groups. Immunohistochemical analyses showed an increase in the capillary density of skeletal muscle in antagomiR-17 condition. In order to determine the gene target and potential pathway involved in the biological effect of antagomiR-17, next generation mRNA sequencing was performed. In conclusion, here we show the potential and underlying molecular mechanism of antagomiR-17 treatment in endothelial cell survival and angiogenesis both in vitro and in vivo.

Increased Skeletal Muscle Capillarization Independently Enhances Insulin Sensitivity in Older Adults After Exercise Training and Detraining.

Intramuscular signaling and glucose transport mechanisms contribute to improvements in insulin sensitivity after aerobic exercise training. This study tested the hypothesis that increases in skeletal muscle capillary density (CD) also contribute to exercise-induced improvements in whole-body insulin sensitivity (insulin-stimulated glucose uptake per unit plasma insulin [M/I]) independent of other mechanisms. The study design included a 6-month aerobic exercise training period followed by a 2-week detraining period to eliminate short-term effects of exercise on intramuscular signaling and glucose transport. Before and after exercise training and detraining, 12 previously sedentary older (65 ± 3 years) men and women underwent research tests, including hyperinsulinemic-euglycemic clamps and vastus lateralis biopsies. Exercise training increased Vo2max (2.2 ± 0.2 vs. 2.5 ± 0.2 L/min), CD (313 ± 13 vs. 349 ± 18 capillaries/mm(2)), and M/I (0.041 ± 0.005 vs. 0.051 ± 0.007 μmol/kg fat-free mass/min) (P < 0.05 for all). Exercise training also increased the insulin activation of glycogen synthase by 60%, GLUT4 expression by 16%, and 5' AMPK-α1 expression by 21%, but these reverted to baseline levels after detraining. Conversely, CD and M/I remained 15% and 18% higher after detraining, respectively (P < 0.05), and the changes in M/I (detraining minus baseline) correlated directly with changes in CD in regression analysis (partial r = 0.70; P = 0.02). These results suggest that an increase in CD is one mechanism contributing to sustained improvements in glucose metabolism after aerobic exercise training.