Muscle Capillary Density Research Articles

Increased cGMP improves microvascular exercise training adaptations independent of endothelial nitric oxide synthase.

Impaired microvascular function is a hallmark of pre-diabetes. With development of atherosclerosis this impaired microvascular function can result in diminished capacity for ambulation and is a risk factor for Type 2 Diabetes. Dynamic changes in vascular tone are determined, in large part, by the eNOS/NO/cGMP axis. We used gain of function of the eNOS/NO/cGMP axis in diet-induced obese (DIO) mice and reduced function in lean mice to test the hypothesis that functionality of this vascular control mechanism parallels the benefits of an exercise training regimen. DIO mice have lower exercise capacity than lean mice and were used for pharmacological gain of function. The PDE-5a inhibitor - sildenafil - increases cGMP and was administered to DIO mice daily. In sedentary mice, we find that sildenafil does not improve exercise capacity. In contrast, it amplifies the microcirculatory effects of exercise training. Sildenafil synergizes with exercise training to improve performance during an incremental exercise test. Improved exercise performance was accompanied by increased skeletal muscle capillary flow velocity and capillary density measured via intravital microscopy. Loss of function was tested in lean mice hemizygous for endothelial cell (EC) specific eNOS creating an EC-eNOS knockdown (KD). EC-eNOS KD decreases capillary density and exercise tolerance in sedentary mice; however, it did not prevent exercise-training induced improvements in endurance capacity. These data show that 1) increasing cGMP with sildenafil enhances microcirculatory function and exercise work tolerance that results from training; 2) eNOS KD does not prevent the microcirculatory or improvements in exercise tolerance with training. PDE-5a inhibitors combined with physical exercise are a potential mechanism for improving ambulation in patients with circulatory limitations.

Exercise training alters skeletal muscle microvascular endothelial cell properties in recent postmenopausal females.

The present study examined and compared the impact of exercise training on redox and molecular properties of human microvascular endothelial cells derived from skeletal muscle biopsies from sedentary recent (RPF, ≤ 5 years as postmenopausal) and late (LPF, ≥ 10 years as postmenopausal) postmenopausal females. Resting skeletal muscle biopsies were obtained before and after 8weeks of intense aerobic exercise training for isolation of microvascular endothelial cells and determination of skeletal muscle angiogenic proteins and capillarisation. The microvascular endothelial cells were analysed for mitochondrial respiration and production of reactive oxygen species (ROS), glycolysis and proteins related to vascular function, redox balance and oestrogen receptors. Exercise training led to a reduced endothelial cell ROS formation (∼50%; P=0.009 and P=0.020 for intact and permeabilized cells (state 3), respectively) in RPF only, with no effect on endothelial mitochondrial capacity in either group. Basal endothelial cell lactate formation was higher (7%; P=0.028), indicating increased glycolysis, after compared to before the exercise training period in RPF only. Baseline endothelial G protein-coupled oestrogen receptor (P = 0.028)and muscle capillarisation (P = 0.028) was lower in LPF than in RPF. Muscle vascular endothelial growth factor protein was higher (32%; P=0.002) following exercise training in LPF only. Exercise training did not influence endothelial cell proliferation or skeletal muscle capillarisation in either group, but the CD31 level in the muscle tissue, indicating endothelial cell content, was higher (>50%; P<0.05) in both groups. In conclusion, 8weeks of intense aerobic exercise training reduces ROS formation and enhances glycolysis in microvascular endothelial cells from RPF but does not induce skeletal muscle angiogenesis. KEY POINTS: Late postmenopausal females have been reported to achieve limited vascular adaptations to exercise training. There is a paucity of data on the effect of exercise training on isolated skeletal muscle microvascular endothelial cells (MMECs). In this study the formation of reactive oxygen species in MMECs was reduced and glycolysis increased after 8weeks of aerobic exercise training in recent but not late postmenopausal females. Late postmenopausal females had lower levels of G protein-coupled oestrogen receptor in MMECs and lower skeletal muscle capillary density at baseline. Eight weeks of intense exercise training altered MMEC properties but did not induce skeletal muscle angiogenesis in postmenopausal females.

Mitochondrial targeted catalase improves muscle strength following arteriovenous fistula creation in mice with chronic kidney disease

Hand dysfunction is a common observation after arteriovenous fistula (AVF) creation for hemodialysis access and has a variable clinical phenotype; however, the underlying mechanism responsible is unclear. Grip strength changes are a common metric used to assess AVF-associated hand disability but has previously been found to poorly correlate with the hemodynamic perturbations post-AVF placement implicating other tissue-level factors as drivers of hand outcomes. In this study, we sought to test if expression of a mitochondrial targeted catalase (mCAT) in skeletal muscle could reduce AVF-related limb dysfunction in mice with chronic kidney disease (CKD). Male and female C57BL/6J mice were fed an adenine-supplemented diet to induce CKD prior to placement of an AVF in the iliac vascular bundle. Adeno-associated virus was used to drive expression of either a green fluorescent protein (control) or mCAT using the muscle-specific human skeletal actin (HSA) gene promoter prior to AVF creation. As expected, the muscle-specific AAV-HSA-mCAT treatment did not impact blood urea nitrogen levels (P = 0.72), body weight (P = 0.84), or central hemodynamics including infrarenal aorta and inferior vena cava diameters (P > 0.18) or velocities (P > 0.38). Hindlimb perfusion recovery and muscle capillary densities were also unaffected by AAV-HSA-mCAT treatment. In contrast to muscle mass and myofiber size which were not different between groups, both absolute and specific muscle contractile forces measured via a nerve-mediated in-situ preparation were significantly greater in AAV-HSA-mCAT treated mice (P = 0.0012 and P = 0.0002). Morphological analysis of the post-synaptic neuromuscular junction uncovered greater acetylcholine receptor cluster areas (P = 0.0094) and lower fragmentation (P = 0.0010) in AAV-HSA-mCAT treated mice. Muscle mitochondrial oxidative phosphorylation was not different between groups, but AAV-HSA-mCAT treated mice had lower succinate-fueled mitochondrial hydrogen peroxide emission compared to AAV-HSA-GFP mice (P < 0.001). In summary, muscle-specific scavenging of mitochondrial hydrogen peroxide significantly improves neuromotor function in mice with CKD following AVF creation.

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.

N-acetylcysteine treatment attenuates hemodialysis access-related limb pathophysiology in mice with chronic kidney disease.

The objective of the present study was to determine if treatment with N-acetylcysteine (NAC) could reduce access-related limb dysfunction in mice. Male and female C57BL6J mice were fed an adenine-supplemented diet to induce chronic kidney disease (CKD) prior to the surgical creation of an arteriovenous fistula (AVF) in the iliac vascular bundle. AVF creation significantly increased peak aortic and infrarenal vena cava blood flow velocities, but NAC treatment had no significant impact, indicating that fistula maturation was not impacted by NAC treatment. Hindlimb muscle and paw perfusion recovery and muscle capillary density in the AVF limb were unaffected by NAC treatment. However, NAC treatment significantly increased the mass of the tibialis anterior (P = 0.0120) and soleus (P = 0.0452) muscles post-AVF. There was a significant main effect of NAC treatment on hindlimb grip strength at postoperative day 12 (POD 12) (P = 0.0003), driven by significantly higher grip strength in both male (P = 0.0273) and female (P = 0.0031) mice treated with NAC. There was also a significant main effect of NAC treatment on the walking speed at postoperative day 12 (P = 0.0447), and post hoc testing revealed an improvement in NAC-treated male mice (P = 0.0091). The area of postsynaptic acetylcholine receptors (P = 0.0263) and motor endplates (P = 0.0240) was also increased by NAC treatment. Interestingly, hindlimb skeletal muscle mitochondrial oxidative phosphorylation trended higher in NAC-treated female mice but was not statistically significant (P = 0.0973). Muscle glutathione levels and redox status were not significantly impacted by NAC treatment in either sex. In summary, NAC treatment attenuated some aspects of neuromotor pathology in mice with chronic kidney disease following AVF creation.NEW & NOTEWORTHY Hemodialysis via autogenous arteriovenous fistula (AVF) is the preferred first-line modality for renal replacement therapy in patients with end-stage kidney disease. However, patients undergoing AVF surgery frequently experience a spectrum of hand disability symptoms postsurgery including weakness and neuromotor dysfunction. Unfortunately, no treatment is currently available to prevent or mitigate these symptoms. Here, we provide evidence that daily N-acetylcysteine supplementation can attenuate some aspects of limb neuromotor function in a preclinical mouse model of AVF.

Endothelial Progenitor Cells and Macrophage Subsets Recruitment in Postischemic Mouse Hind Limbs

Introduction: Peripheral arterial disease (PAD) occurs from atherosclerotic obstruction of arteries in the lower extremities. Restoration of perfusion requires angiogenesis and arteriogenesis through migration and differentiation of endothelial progenitor cells (EPCs) and macrophages at the site of injury. The time of recruitment has not been fully investigated. In this study, we investigated the infiltration of these cells in murine hind limb ischemia (HLI) model of PAD. Methods: EPCs and M1-like and M2-like macrophages from ischemic skeletal muscles were quantified by flow cytometry at day-0, 1, 3, 7, and 14 post-HLI. Results: The abundance of EPCs increased from day 1 and was highest on day 7 until day 14. M1-like population similarly increased and was highest on day 14 during the experiment. M2-like population was significantly greater than M1-like at baseline but surpassed the highest value of M1-like by day 7 during the experiment. Muscle regeneration and capillary density also increased and were highest at days 3 and 7, respectively, during the experiment. All mice achieved near full perfusion recovery by day 14. Conclusion: Thus, we observed a gradual increase in the percentage of EPC’s and this was temporally paralleled with initial increase in M1-like followed by sustained increased in M2-like macrophages and perfusion recovered post-HLI.

Effects of projected end-of-century temperature on the muscle development of neonate epaulette sharks, Hemiscyllium ocellatum

Epaulette sharks (Hemiscyllium ocellatum) inhabit shallow tropical habitats with elevated and fluctuating temperatures. Yet, according to global climate change projections, water temperatures in these habitats will rise beyond current cyclical variability, warranting further studies incorporating chronically elevated temperature exposure in this species. This study examined the differences in skeletal muscle morphological and metabolic properties in neonate epaulette sharks exposed to their current-day ambient (27 °C) or projected end-of-century (31 °C) habitat temperatures throughout embryonic and neonatal development. Metrics of skeletal muscle, such as muscle fiber size and density, nuclear density, and satellite cell density, were used to assess the relative contribution of hypertrophic and hyperplastic growth processes. Capillary density was measured as a proxy for peripheral oxygen supply to muscle tissue. At 31 °C, sharks hatched earlier, but were similar in body size 60 days post-hatch. Muscle fiber size, nuclear density, and capillary density were similar between temperature regimes. However, fiber density was lower, satellite cell density was higher, and fibers associated with satellite cells were smaller in sharks reared at 31 °C. These results suggest that elevated temperature may impair or slow satellite cell fusion to existing fibers and new fiber formation. To assess potential metabolic and developmental consequences of elevated temperatures, oxidative damage (2,4-DNPH, 8-OHdG, 4-HNE), protein degradation (Ubiquitin, LC3B, Hsp70), and muscle differentiation (Myf5, Myogenin) markers were measured. Protein carbonylation was higher at elevated temperatures, suggesting that warmer incubation temperatures at early life stages may result in oxidative damage accrual. However, protein degradation and muscle differentiation markers did not differ. These results suggest that projected end-of-century temperatures may alter muscle growth and metabolism in tropical shark species with potential consequences to shark growth and fitness.

Endothelial SIRT-1 has a critical role in the maintenance of capillarization in brown adipose tissue

Brown adipose tissue (BAT) has critical roles in thermogenesis and systemic metabolism. Capillary rarefaction was reported to develop in BAT with dietary obesity, and previous studies showed that suppression of vascular endothelial growth factor A (VEGF-A) reduced capillary density in BAT, promoting the functional decline of this organ. Capillarization is regulated through the balance between angiogenesis and vasculogenesis on the one hand and apoptosis of endothelial cells (ECs) on the other; however, the role of EC apoptosis in BAT remained to be explored. In studies testing the role of boysenberry polyphenols (BoyP) in BAT, we found that BoyP decreased EC apoptosis, enhanced capillarization in BAT, and ameliorated dietary BAT dysfunction, which was associated with the upregulation of nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin 1 (SIRT-1) in ECs. Our studies suggest that EC SIRT-1 would be one of the potential targets of BoyP that contributes to BAT capillarization and function.

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&amp;lt;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)

Extreme Variations in Muscle Fiber Composition Enable Detection of Insulin Resistance and Excessive Insulin Secretion.

Muscle fiber composition is associated with peripheral insulin action. We investigated whether extreme differences in muscle fiber composition are associated with alterations in peripheral insulin action and secretion in young, healthy subjects who exhibit normal fasting glycemia and insulinemia. Relaxation time following a tetanic contraction was used to identify subjects with a high or low expression of type I muscle fibers: group 1 (n = 11), area occupied by type I muscle fibers = 61.0 ± 11.8%, and group 2 (n = 8), type I area = 36.0 ± 4.9% (P < 0.001). Biopsies were obtained from the vastus lateralis muscle and analyzed for mitochondrial respiration on permeabilized fibers, muscle fiber composition, and capillary density. An intravenous glucose tolerance test was performed and indices of glucose tolerance, insulin sensitivity, and secretion were determined. Glucose tolerance was similar between groups, whereas whole-body insulin sensitivity was decreased by ~50% in group 2 vs group 1 (P = 0.019). First-phase insulin release (area under the insulin curve during 10 minutes after glucose infusion) was increased by almost 4-fold in group 2 vs group 1 (P = 0.01). Whole-body insulin sensitivity was correlated with percentage area occupied by type I fibers (r = 0.54; P = 0.018) and capillary density in muscle (r = 0.61; P = 0.005) but not with mitochondrial respiration. Insulin release was strongly related to percentage area occupied by type II fibers (r = 0.93; P < 0.001). Assessment of muscle contractile function in young healthy subjects may prove useful in identifying individuals with insulin resistance and enhanced glucose-stimulated insulin secretion prior to onset of clinical manifestations.

Probiotic Strains Isolated from an Olympic Woman's Weightlifting Gold Medalist Increase Weight Loss and Exercise Performance in a Mouse Model.

Obesity is a worldwide health problem. Calorie-restricted diets constitute a common intervention for treating obesity. However, an improper calorie-restricted diet can lead to malnutrition, fatigue, poor concretion, muscle loss, and reduced exercise performance. Probiotics have been introduced as an alternative treatment for obesity. In the present study, we tested the weight loss and exercise performance enhancement effectiveness of probiotic strains of different origins, including four isolated from an Olympic weightlifting gold medalist (Bifidobacterium longum subsp. longum OLP-01, Lactobacillus plantarum PL-02, Lactobacillus salivarius subsp. salicinius SA-03, and Lactococcus lactis subsp. lactis LY-66). A high-fat diet (HFD) was used to induce obesity in 16 groups of mice (n = 8/group). The mice were administered probiotic supplements at a dosage of 4.1 × 109 CFU/kg/day for 10 weeks. All probiotic supplementation groups showed a significant reduction in body weight and fat mass compared with the HFD group. TYCA06, CS-773, BLI-02, PL-02, bv-77, and OLP-01 were the most effective in facilitating weight loss and fat reduction, which may be due to fatty-acid absorbing activity. PL-02, LY-66, TYCA06, CS-773, and OLP-01 elevated the animals’ grip strength and exhaustive running duration. PL-02, LY-66, and OLP-01 increased tissue glycogen (liver and muscle) levels and muscle capillary density and reduced blood lactate production levels after exercise. In conclusion, OLP-01, PL-02, LY-66, TYCA06, and CS-773 were highly effective in enhancing weight loss and exercise performance. This study should be repeated on humans in the future to further confirm the findings.

Endothelial Piezo1 sustains muscle capillary density and contributes to physical activity.

Piezo1 forms mechanically activated nonselective cation channels that contribute to endothelial response to fluid flow. Here we reveal an important role in the control of capillary density. Conditional endothelial cell–specific deletion of Piezo1 in adult mice depressed physical performance. Muscle microvascular endothelial cell apoptosis and capillary rarefaction were evident and sufficient to account for the effect on performance. There was selective upregulation of thrombospondin-2 (TSP2), an inducer of endothelial cell apoptosis, with no effect on TSP1, a related important player in muscle physiology. TSP2 was poorly expressed in muscle endothelial cells but robustly expressed in muscle pericytes, in which nitric oxide (NO) repressed the Tsp2 gene without an effect on Tsp1. In endothelial cells, Piezo1 was required for normal expression of endothelial NO synthase. The data suggest an endothelial cell–pericyte partnership of muscle in which endothelial Piezo1 senses blood flow to sustain capillary density and thereby maintain physical capability.

The Impact of Vegan and Vegetarian Diets on Physical Performance and Molecular Signaling in Skeletal Muscle.

Muscular adaptations can be triggered by exercise and diet. As vegan and vegetarian diets differ in nutrient composition compared to an omnivorous diet, a change in dietary regimen might alter physiological responses to physical exercise and influence physical performance. Mitochondria abundance, muscle capillary density, hemoglobin concentration, endothelial function, functional heart morphology and availability of carbohydrates affect endurance performance and can be influenced by diet. Based on these factors, a vegan and vegetarian diet possesses potentially advantageous properties for endurance performance. Properties of the contractile elements, muscle protein synthesis, the neuromuscular system and phosphagen availability affect strength performance and can also be influenced by diet. However, a vegan and vegetarian diet possesses potentially disadvantageous properties for strength performance. Current research has failed to demonstrate consistent differences of performance between diets but a trend towards improved performance after vegetarian and vegan diets for both endurance and strength exercise has been shown. Importantly, diet alters molecular signaling via leucine, creatine, DHA and EPA that directly modulates skeletal muscle adaptation. By changing the gut microbiome, diet can modulate signaling through the production of SFCA.

Lycopene ameliorates insulin resistance and increases muscle capillary density in aging via activation of SIRT1

Lycopene (Ly) is a kind of hydrocarbon, which belongs to the family of tetraterpene carotene and exists in red fruits and vegetables. The decrease of capillary density and blood flow with age is a significant reason for the increase of mortality and morbidity. Herein, our study aims to explore the effects of Ly (a bioactive food compound) on vascular aging in vitro and in vivo and its potential mechanisms. The cytological results showed that Ly could promote the proliferation of human umbilical vein endothelial cell (HUVECs) and enhance the ability of HUVECs to form capillary-like structures. Furthermore, the expression of SIRT1 in aged HUVECs was up-regulated. In vivo, aging rats showed signs of insulin resistance and blood vessel damage. Additionally, the capillary density and blood flow were reduced during the vascular aging process in both D‐gal‐induced and naturally aging muscle. However, when Ly was given, these conditions could be reversed. Simultaneously, the contents of ATP, lactic acid and pyruvic acid were determined, and it was found that Ly could promote angiogenesis by increasing the utilization rate of glucose and promoting energy metabolism. Finally, in the insulin resistance cell model, we knocked down the SIRT1 and administrated with Ly, and found that it couldn't restore insulin transdution. In conclusion, all the data in this study demonstrate that Ly could reactivate SIRT1 and improve insulin resistance, which was a reversible cause of vascular aging.

Endothelial Insulin Receptors Promote VEGF-A Signaling via ERK1/2 and Sprouting Angiogenesis.

Endothelial insulin receptors (Insr) promote sprouting angiogenesis, although the underpinning cellular and molecular mechanisms are unknown. Comparing mice with whole-body insulin receptor haploinsufficiency (Insr+/-) against littermate controls, we found impaired limb perfusion and muscle capillary density after inducing hind-limb ischemia; this was in spite of increased expression of the proangiogenic growth factor Vegfa. Insr+/- neonatal retinas exhibited reduced tip cell number and branching complexity during developmental angiogenesis, which was also found in separate studies of mice with endothelium-restricted Insr haploinsufficiency. Functional responses to vascular endothelial growth factor A (VEGF-A), including in vitro angiogenesis, were also impaired in aortic rings and pulmonary endothelial cells from Insr+/- mice. Human umbilical vein endothelial cells with shRNA-mediated knockdown of Insr also demonstrated impaired functional angiogenic responses to VEGF-A. VEGF-A signaling to Akt and endothelial nitric oxide synthase was intact, but downstream signaling to extracellular signal-reduced kinase 1/2 (ERK1/2) was impaired, as was VEGF receptor-2 (VEGFR-2) internalization, which is required specifically for signaling to ERK1/2. Hence, endothelial insulin receptors facilitate the functional response to VEGF-A during angiogenic sprouting and are required for appropriate signal transduction from VEGFR-2 to ERK1/2.

CD146+Mesenchymal stem cells treatment improves vascularization, muscle contraction and VEGF expression, and reduces apoptosis in rat ischemic hind limb

Peripheral arterial disease (PAD) is an increasingly common narrowing of the peripheral arteries that can lead to lower limb ischemia, muscle weakness and gangrene. Surgical vein or arterial grafts could improve PAD, but may not be suitable in elderly patients, prompting research into less invasive approaches. Mesenchymal stem cells (MSCs) have been proposed as potential therapy, but their effectiveness and underlying mechanisms in limb ischemia are unclear. We tested the hypothesis that treatment with naive MSCs (nMSCs) or MSCs expressing CD146 (CD146+MSCs) could improve vascularity and muscle function in rat model of hind-limb ischemia. Sixteen month old Sprague-Dawley rats were randomly assigned to 4 groups: sham-operated control, ischemia, ischemia + nMSCs and ischemia+CD146+MSCs. After 4 weeks of respective treatment, rat groups were assessed for ischemic clinical score, Tarlov score, muscle capillary density, TUNEL apoptosis assay, contractile force, and vascular endothelial growth factor (VEGF) mRNA expression. CD146+MSCs showed greater CD146 mRNA expression than nMSCs. Treatment with nMSCs or CD146+MSCs improved clinical and Tarlov scores, muscle capillary density, contractile force and VEGF mRNA expression in ischemic limbs as compared to non-treated ischemia group. The improvements in muscle vascularity and function were particularly greater in ischemia+CD146+MSCs than ischemia + nMSCs group. TUNEL positive apoptotic cells were least abundant in ischemia+CD146+MSCs compared with ischemia + nMSCs and non-treated ischemia groups. Thus, MSCs particularly those expressing CD146 improve vascularity, muscle function and VEGF expression and reduce apoptosis in rat ischemic limb, and could represent a promising approach to improve angiogenesis and muscle function in PAD.

Pioglitazone does not synergize with mirabegron to increase beige fat or further improve glucose metabolism.

BACKGROUNDBeige and brown adipose tissue (BAT) are associated with improved metabolic homeostasis. We recently reported that the β3-adrenergic receptor agonist mirabegron induced beige adipose tissue in obese insulin-resistant subjects, and this was accompanied by improved glucose metabolism. Here we evaluated pioglitazone treatment with a combination pioglitazone and mirabegron treatment and compared these with previously published data evaluating mirabegron treatment alone. Both drugs were used at FDA-approved dosages.METHODSWe measured BAT by PET CT scans, measured beige adipose tissue by immunohistochemistry, and comprehensively characterized glucose and lipid homeostasis and insulin sensitivity by euglycemic clamp and oral glucose tolerance tests. Subcutaneous white adipose tissue, muscle fiber type composition and capillary density, lipotoxicity, and systemic inflammation were evaluated by immunohistochemistry, gene expression profiling, mass spectroscopy, and ELISAs.RESULTSTreatment with pioglitazone or the combination of pioglitazone and mirabegron increased beige adipose tissue protein marker expression and improved insulin sensitivity and glucose homeostasis, but neither treatment induced BAT in these obese subjects. When the magnitude of the responses to the treatments was evaluated, mirabegron was found to be the most effective at inducing beige adipose tissue. Although monotherapy with either mirabegron or pioglitazone induced adipose beiging, combination treatment resulted in less beiging than either alone. The 3 treatments also had different effects on muscle fiber type switching and capillary density.CONCLUSIONThe addition of pioglitazone to mirabegron treatment does not enhance beiging or increase BAT in obese insulin-resistant research participants.TRIAL REGISTRATIONClinicalTrials.gov NCT02919176.FUNDINGNIH DK112282 and P20GM103527 and Clinical and Translational Science Awards grant UL1TR001998.

Aerobic exercise leads to upregulation of Mir-126 and angiogenic signaling in the heart tissue of diabetic rats

ObjectiveMicroRNA-126 plays a critical role in promoting cardiac angiogenesis. Aerobic exercise has also cardioprotective effects by upregulating angiogenic factors. The present study aimed to evaluate the impact of aerobic exercise on cardiac expression of mir-126 and angiogenic signaling pathways. MethodsThirty-two male Wistar rats (180–240 g) were randomly divided into four diabetic control, healthy control, healthy exercise, and diabetic exercise groups. Diabetes was induced by intraperitoneal injection of Streptozotocin (STZ) 50 mg/kg (with citrate buffer) and nicotinamide 110 mg/kg (with saline). Aerobic training groups were performing on a rodent treadmill with an intensity of 25 m/min, 5° gradients, and 30 min each session for 8 weeks. The control groups were placed on a stationary treadmill without training. The heart of the rats was taken and the expression level of cardiac mir-126 was evaluated by Real-time PCR. The protein levels of raf1, spred1, and VEGF were measured using ELISA. Immunohistochemistry was used to measure cardiac muscle capillary density. ResultsThe results showed that diabetes induced by STZ disrupted angiogenesis signaling through the reduction of mir-126, raf1, and VEGF, while an increase in spred1 protein level (p = 0.01 for all). However, 8 weeks of aerobic exercise (in healthy animals) significantly increased mir-126 expression, and raf1 and VEGF proteins (p = 0.05), while significantly decreased spred1 protein (p = 0.05). Also, 8 weeks of exercise training in diabetic rats might improve the cardiac tissue by increasing mir-126 and angiogenic signaling pathways. ConclusionThe results of this study suggest that aerobic exercise training may prevent negative effects of diabetes on heart tissue by improving angiogenic pathway. Therefore, aerobic exercise may be a good strategy for coping with cardiovascular cell damage of diabetes.

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.