Skeletal Muscle Microvasculature Research Articles

Determinants of the diminished exercise capacity in patients with chronic obstructive pulmonary disease: looking beyond the lungs.

Peak oxygen uptake, a primary determinant of prognosis, mortality and quality of life, is diminished in patients with chronic obstructive pulmonary disease (COPD), with mounting evidence supporting an important role for peripheral dysfunction, particularly within skeletal muscle. In patients with severe COPD and activity-matched controls, muscle oxygen transport and utilization were assessed at peak effort during single-leg knee-extensor exercise (KE), where ventilation is assumed to be submaximal. This strategy removes ventilation as the major constraint to exercise capacity in COPD, allowing maximal muscle function to be attained and evaluated. During maximal KE, both convective arterial oxygen delivery to the skeletal muscle microvasculature and subsequent diffusive oxygen delivery to the mitochondria were diminished in patients with COPD compared to control subjects. These findings emphasize the importance of factors, beyond the lungs, that influence exercise capacity in this patient population and may, ultimately, influence the prognosis, mortality and quality of life for patients with COPD. Peak oxygen uptake ( ), a primary determinant of prognosis, mortality and quality of life, is diminished in patients with chronic obstructive pulmonary disease (COPD). Mounting evidence supports an important role of the periphery, particularly skeletal muscle, in the diminished with COPD. However, the peripheral determinants of have not been comprehensively assessed in this cohort. Thus, the hypothesis was tested that both muscle convective and diffusive oxygen (O2 ) transport, and therefore skeletal muscle peak O2 uptake ( ), are diminished in patients with COPD compared to matched healthy controls, even when ventilatory limitations (i.e. attainment of maximal ventilation) are minimized by using small muscle mass exercise. Muscle O2 transport and utilization were assessed at peak exercise from femoral arterial and venous blood samples and leg blood flow (by thermodilution) in eight patients with severe COPD (forced expiratory volume in 1s (FEV1 )±SEM=0.9±0.1l, 30% of predicted) and eight controls during single-leg knee-extensor exercise. Both muscle convective O2 delivery (0.44±0.06vs. 0.69±0.07lmin-1 , P<0.05) and muscle diffusive O2 conductance (6.6±0.8vs. 10.4±0.9mlmin-1 mmHg-1 , P<0.05) were ∼1/3 lower in patients with COPD than controls, resulting in an attenuated in the patients (0.27±0.04vs. 0.42±0.05lmin-1 , P<0.05). When cardiopulmonary limitations to exercise are minimized, the convective and diffusive determinants of , at the level of the skeletal muscle, are greatly attenuated in patients with COPD. These findings emphasize the importance of factors, beyond the lungs, that may ultimately influence this population's prognosis, mortality and quality of life.

Beneficial effects of endurance exercise training on skeletal muscle microvasculature in sickle cell disease patients

AbstractSickle cell disease (SCD) is a genetic hemoglobinopathy leading to 2 major clinical manifestations: severe chronic hemolytic anemia and iterative vaso-occlusive crises. SCD is also accompanied by profound muscle microvascular remodeling. The beneficial effects of endurance training on microvasculature are widely known. The aim of this study was to evaluate the effects of an endurance training program on microvasculature of skeletal muscle in SCD patients. A biopsy of the vastus lateralis muscle and submaximal incremental exercise was performed before and after the training period. Of the 40 randomized SCD patients, complete data sets from 32 patients were obtained. The training group (n = 15) followed a personalized moderate-intensity endurance training program, while the nontraining (n = 17) group maintained a normal lifestyle. Training consisted of three 40-minute cycle ergometer exercise sessions per week for 8 weeks. Histological analysis highlighted microvascular benefits in the training SCD patients compared with nontraining patients, including increases in capillary density (P = .003), number of capillaries around a fiber (P = .015), and functional exchange surface (P < .0001). Conversely, no significant between-group difference was found in the morphology of capillaries. Indexes of physical ability also improved in the training patients. The moderate-intensity endurance exercise training program improved the muscle capillary network and partly reversed the microvascular defects commonly observed in skeletal muscle of SCD patients. This trial was registered at www.clinicaltrials.gov as #NCT02571088.

High-Fat Overfeeding Impairs Peripheral Glucose Metabolism and Muscle Microvascular eNOS Ser1177 Phosphorylation.

The mechanisms responsible for dietary fat-induced insulin resistance of skeletal muscle and its microvasculature are only partially understood. To determine the impact of high-fat overfeeding on postprandial glucose fluxes, muscle insulin signaling, and muscle microvascular endothelial nitric oxide synthase (eNOS) content and activation. Fifteen non-obese volunteers consumed a high-fat (64%) high-energy (+47%) diet for 7 days. Experiments were performed before and after the diet. Stable isotope tracers were used to determine glucose fluxes in response to carbohydrate plus protein ingestion. Muscle insulin signaling was determined as well as the content and activation state of muscle microvascular eNOS. High-fat overfeeding impaired postprandial glycemic control as demonstrated by higher concentrations of glucose (+11%; P = 0.004) and insulin (+19%; P = 0.035). Carbohydrate plus protein ingestion suppressed endogenous glucose production to a similar extent before and after the diet. Conversely, high-fat overfeeding reduced whole-body glucose clearance (-16%; P = 0.021) and peripheral insulin sensitivity (-26%; P = 0.006). This occurred despite only minor alterations in skeletal muscle insulin signaling. High-fat overfeeding reduced eNOS content in terminal arterioles (P = 0.017) and abolished the increase in eNOS Ser1177 phosphorylation that was seen after carbohydrate plus protein ingestion. High-fat overfeeding impaired whole-body glycemic control due to reduced glucose clearance, not elevated endogenous glucose production. The finding that high-fat overfeeding abolished insulin-mediated eNOS Ser1177 phosphorylation in the terminal arterioles suggests that impairments in the vasodilatory capacity of the skeletal muscle microvasculature may contribute to early dietary fat-induced impairments in glycemic control.

Skeletal muscle microvasculature response to β-adrenergic stimuli is diminished with cardiac surgery

BackgroundCardiac surgery and cardiopulmonary bypass are associated with alterations in blood pressure in the perioperative period, which, if uncontrolled, can result in end organ damage or dysfunction. Microvessels, significant contributors to blood pressure, both in the myocardium and peripheral skeletal muscle, have diminished responsiveness to major mediators of vascular tone, including thromboxane and serotonin after cardiopulmonary bypass. Responsiveness of these vessels to β-adrenergic stimulation, a major mediator of vascular tone, has not yet been studied. In this report, we investigated the role of β-adrenergic receptors in vascular tone regulation in human skeletal muscle microvessels before and after β-adrenergic stimulation. MethodsSkeletal muscle microvessels were isolated from patients undergoing cardiac surgery before and after cardiopulmonary bypass. Vessels were exposed in an ex vivo model to the β-adrenergic agonist isoproterenol, or the direct adenylyl cyclase activator, forskolin, and the selective β-receptor antagonist ICI18.551 hydrochloride plus isoproterenol. Immunofluorescence of β receptors and Western blotting were also performed. ResultsMicrovessels showed diminished responsiveness to isoproterenol (10–6 to 10–4M) after cardiopulmonary bypass (n = 8/group, P = .01). Pretreatment with the selective β-2 blocker ICI18.551 (10–6M) prevented isoproterenol-induced microvascular relaxation (P = .001). Forskolin-induced relaxation response was also significantly diminished after cardiopulmonary bypass (n = 4/group, P < .05 versus before cardiopulmonary bypass). No significant changes in the total protein expression of β-1, β-2, and β-3 receptors were detected by western blotting or immunofluorescence. ConclusionMicrovessels isolated from human skeletal muscle show diminished responsiveness to isoproterenol and its downstream activator forskolin after cardiopulmonary bypass, suggesting there is an alteration in β-adrenergic receptor responsive in adenylate cyclase. The relaxation response to isoproterenol was via activation β-2 receptors without changes in β-adrenergic receptor abundance.

1979-P: GLP-1 Recruits Skeletal Muscle Microvasculature without Impacting Glucose Uptake or Protein Metabolism in Older Men during Postabsorptive Insulin Clamps

Introduction: Ageing muscle is associated with impaired blood flow, “anabolic resistance” to dietary protein, and impaired glucose uptake - culminating in muscle atrophy and insulin resistance. Since GLP-1 has been shown to recruit muscle microvasculature, and due to the presence of its receptors in local tissue(s) (e.g., endothelium, myocytes) we hypothesized that GLP-1 infusions in older people, would positively impact muscle microcirculation, protein and/or glucose metabolism - in an insulin-independent fashion. Methods: We studied seven older men (∼70±1 y, mean±SEM) in a cross-over design. Baseline measures were taken (first 3 h) prior to insulin and glucose clamps where participants received co-infusions of octreotide, Vamin 14-EF (containing essential amino acids and dextrose) - with or without GLP-1, over the next 3 h. Four muscle biopsies were taken over 90-120 minute intervals. Muscle protein turnover was quantified via A-V balance, and direct incorporation of 13C6 phenylalanine via mass spectrometric techniques. Plasma GLP-1 and insulin were quantified via Milliplex Map, ELISA. Muscle microvascular flow was assessed via contrast enhanced ultrasound. Leg glucose uptake was determined via A-V balance. Results: GLP-1 increased microvascular flow in +GLP-1 vs. -GLP-1 conditions (increase vs. without GLP-1 infusion: 7.2±4.7 vs. 0.40±0.26-fold, p=0.009) while leg glucose uptake was unchanged. Muscle proteolysis (nb. no effects on synthesis) was equally suppressed vs. basal with GLP-1 (32.1±5.3 vs. 67.4±3.2 nmol.100 g leg-1.min-1, p&amp;lt;0.05) and without GLP-1 (32.77±6.1 vs. 57.9±5.1 nmol.100 g leg-1.min-1 p&amp;lt;0.05, respectively). Conclusion: GLP-1 recruits muscle microcirculation independent of insulin. A lack of impact of GLP-1 on muscle glucose uptake or protein metabolism, could reflect a requirement for fed-state insulin levels. GLP-1 may hold therapeutic value for muscle microvascular health. Disclosure H. Abdulla: None. B.E. Phillips: None. M.C. Limb: None. D.J. Wilkinson: None. T. Jandova: None. J. Cegielski: None. J.J. Bass: None. D. Rankin: None. J.S. Lewis: None. J. Williams: None. K. Smith: None. P.J. Atherton: None. I.R. Idris: None. Funding Medical Research Council UK; Arthritis Research UK

Arterial Network Geometric Characteristics and Regulation of Capillary Blood Flow in Hamster Skeletal Muscle Microcirculation.

This study was aimed to characterize the geometric arrangement of hamster skeletal muscle arteriolar networks and to assess the in vivo rhythmic diameter changes of arterioles to clarify regulatory mechanisms of the capillary perfusion. The experimental study was carried out in male Syrian hamsters implanted with a plastic chamber in the dorsum skin under pentobarbital anesthesia. The skeletal muscle microvessels were visualized by fluorescence microscopy. The vessel diameters, lengths and the rhythmic diameter changes of arterioles were analyzed with computer-assisted techniques. The arterioles were classified according to a centripetal ordering scheme. In hamster skeletal muscle microvasculature the terminal branchings, differentiated in long and short terminal arteriolar trees (TATs), originated from anastomotic vessels, defined “arcading” arterioles. The long TATs presented different frequencies along the branching vessels; order 4 arterioles had frequencies lower than those observed in the order 3, 2, and 1 vessels. The short TAT order 3 arterioles, directly originating from “arcading” parent vessels, showed a frequency dominating all daughter arterioles. The amplitude of diameter variations in larger vessels was in the range 30–40% of mean diameter, while it was 80–100% in order 3, 2, and 1 vessels. Therefore, the complete constriction of arterioles, caused an intermittent capillary blood perfusion. L-arginine or papaverine infusion caused dilation of arterioles and transient disappearing of vasomotion waves and induced perfusion of all capillaries spreading from short and long TAT arrangements. Therefore, the capillary blood flow was modulated by changes in diameter of terminal arterioles penetrating within the skeletal muscle fibers, facilitating redistribution of blood flow according to the metabolic demands of tissues.

Arteriolar and capillary responses to CO2 and H+ in hamster skeletal muscle microvasculature: Implications for active hyperemia

We hypothesized that CO2 and H+ stimulate capillaries and arterioles to produce local and conducted vasodilations required to coordinate the distribution of blood flow to contracting skeletal muscle fibers. CO2 and H+ independently and in combination were applied to 2A arterioles (first branch order from the 1A feed arteriole) and capillaries of the in situ, blood-perfused hamster cremaster muscle. The resulting local and conducted vasodilations were measured. H+ (pH: 7.2-6.6) and CO2 (5% and 10%) applied to the vascular network induced 2A arteriolar vasodilations, while 15% CO2 produced vasoconstriction. Localized application of H+ produced 2A arteriolar vasodilation, while 15% CO2 resulted in a variable response. Simultaneous application of CO2 and H+ did not result in the predicted additive effects. Application of CO2 and H+ alone or combined on arterioles or capillaries did not induce a conducted response. CO2 and H+ produce arteriolar vasodilation but, critically, cannot stimulate the spread of vasodilation throughout the network, thus limiting their ability to coordinating blood flow to contracting skeletal muscle fibers. Given their potential for interaction, the importance of CO2 and H+ may lie in their ability to modify the effects of other vasodilators.

Deoxy[Hb+Mb] increases consistently up to peak oxygen consumption in elite cyclists

This study aimed to compare deoxygenation in the vastus lateralis in the skeletal muscle microvasculature of nine world-class/elite (WC/E) vs. thirty-one well-trained/trained (T) cyclists by near infrared spectroscopy (NIRS) during a maximal aerobic power (MAP) test. Interaction analysis showed that 1st degree polynomial trends across % peak oxygen consumption (%VO2peak) differed significantly between WC/E and T cyclists on deoxygenated hemoglobin-myoglobin (Δ[HHb]) and on total hemoglobin-myoglobin (Δ[THb]) from 70 to 100% of VO2peak. These results show that the linear slopes of these trends were different. Near-plateau of Δ[HHb] was observed in T cyclists, whereas a consistent increase up to VO2peak was seen in WC/E cyclists. The Δ[THb] curve declined in T cyclists suggesting that blood volume in the vastus lateralis microcirculation decreases from 70 to 100% of VO2peak whereas it increases slowly but continuously to 95% of VO2peak in WC/E cyclists. The results of this study showed that deoxygenation in the vastus lateralis increases consistently up to VO2peak in WC/E cyclists and seems to be influenced by blood volume in the microcirculation.

Glucagon-Like Peptide-1 (GLP-1) Increases Skeletal and Cardiac Muscle, Microvascular Perfusion, and Improves Metabolic Insulin Action in Obese Humans

Obesity is associated with metabolic and microvascular insulin resistance. The latter is characterized by impaired insulin-mediated microvascular recruitment. GLP-1 engenders vasodilation in addition to its glycemic actions. At physiological concentrations, GLP-1 acutely recruits skeletal and cardiac muscle microvasculature in healthy humans and this action is preserved in the insulin resistant rodents. To examine whether GLP-1 recruits microvasculature and improves insulin’s action in obese humans, 15 obese adults (age 27 ± 2.2, BMI 33.7 ± 1.2) were studied thrice at random order after an overnight fast. Subjects received an intravenous infusion of GLP-1 (1.2 pmol/kg/min) for 150 mins or an intravenous infusion of either normal saline or GLP-1 (1.2 pmol/kg/min) for 150 mins with a euglycemic insulin clamp (1 mU/kg/min) superimposed over the last 120 mins. Skeletal and cardiac muscle microvascular blood volume (MBV), flow velocity (MFV) and blood flow (MBF) were determined at 0, 30 and 150 min. Steady-state glucose infusion rate (GIR) was calculated. Insulin infusion alone did not change MBV or MBF in either skeletal or cardiac muscle, confirming the presence of microvascular insulin resistance. GLP-1 infusion alone increased MBV by ∼30% and ∼40% in skeletal and cardiac muscle respectively (p&amp;lt;0.05) without affecting MFV, leading to a significant increase in MBF in both skeletal and cardiac muscle. Superimposition of insulin infusion to GLP-1 infusion did not further increase MBV or MBF in either skeletal or cardiac muscle, but raised the steady-state GIR from 4.1± 0.4 to 5.0 ± 0.5 mg/kg/min (p&amp;lt;0.05). We conclude that in obese humans with microvascular insulin resistance GLP-1's vasodilatory actions are preserved in both skeletal and cardiac muscle microvasculature. This may contribute to improving metabolic responses to insulin and decreasing cardiovascular morbidity and mortality associated with obesity and diabetes. Disclosure N. Wang: None. A. Tan: None. L. Jahn: None. L. Hartline: None. K.W. Aylor: None. E. Barrett: None. Z. Liu: None.

Altered mitochondrial bioenergetics and ultrastructure in the skeletal muscle of young adults with type 1 diabetes.

A comprehensive assessment of skeletal muscle ultrastructure and mitochondrial bioenergetics has not been undertaken in individuals with type 1 diabetes. This study aimed to systematically assess skeletal muscle mitochondrial phenotype in young adults with type 1 diabetes. Physically active, young adults (men and women) with type 1 diabetes (HbA1c 63.0 ± 16.0mmol/mol [7.9% ± 1.5%]) and without type 1 diabetes (control), matched for sex, age, BMI and level of physical activity, were recruited (n = 12/group) to undergo vastus lateralis muscle microbiopsies. Mitochondrial respiration (high-resolution respirometry), site-specific mitochondrial H2O2 emission and Ca2+ retention capacity (CRC) (spectrofluorometry) were assessed using permeabilised myofibre bundles. Electron microscopy and tomography were used to quantify mitochondrial content and investigate muscle ultrastructure. Skeletal muscle microvasculature was assessed by immunofluorescence. Mitochondrial oxidative capacity was significantly lower in participants with type 1 diabetes vs the control group, specifically at Complex II of the electron transport chain, without differences in mitochondrial content between groups. Muscles of those with type 1 diabetes also exhibited increased mitochondrial H2O2 emission at Complex III and decreased CRC relative to control individuals. Electron tomography revealed an increase in the size and number of autophagic remnants in the muscles of participants with type 1 diabetes. Despite this, levels of the autophagic regulatory protein, phosphorylated AMP-activated protein kinase (p-AMPKαThr172), and its downstream targets, phosphorylated Unc-51 like autophagy activating kinase 1 (p-ULK1Ser555) and p62, was similar between groups. In addition, no differences in muscle capillary density or platelet aggregation were observed between the groups. Alterations in mitochondrial ultrastructure and bioenergetics are evident within the skeletal muscle of active young adults with type 1 diabetes. It is yet to be elucidated whether more rigorous exercise may help to prevent skeletal muscle metabolic deficiencies in both active and inactive individuals with type 1 diabetes.

Sympathoexcitation constrains vasodilation in the human skeletal muscle microvasculature during postocclusive reactive hyperemia.

We used diffuse correlation spectroscopy to investigate sympathetic vasoconstriction, local vasodilation, and integration of these two responses in the skeletal muscle microvasculature of 20 healthy volunteers. Diffuse correlation spectroscopy probes were placed on the flexor carpi radialis muscle or vastus lateralis muscle, and a blood flow index was derived continuously. We measured hemodynamic responses during sympathoexcitation induced by forehead cooling, after which the effects of the increased sympathetic tone on vasodilatory responses during postocclusive reactive hyperemia (PORH) were examined. PORH was induced by releasing arterial occlusion (3 min) in an arm or leg. To increase sympathetic tone during PORH, forehead cooling was begun 60 s before the occlusion release and ended 60 s after the release. During forehead cooling, mean arterial pressure rose significantly and was sustained at an elevated level. Significant vasoconstriction and decreases in blood flow index followed by gradual blunting of the vasoconstriction also occurred. The time course of these responses is in good agreement with previous observations in animals. The acute sympathoexcitation diminished the peak vasodilation during PORH only in the vastus lateralis muscle, but it hastened the decline in vasodilation after the peak in both the flexor carpi radialis muscle and vastus lateralis muscle. Consequently, the total vasodilatory response assessed as the area of the vascular conductance during the first minute of PORH was significantly diminished in both regions. We conclude that, in humans, the integrated effects of sympathetic vasoconstriction and local vasodilation have an important role in vascular regulation and control of perfusion in the skeletal muscle microcirculation. NEW & NOTEWORTHY We used diffuse correlation spectroscopy to demonstrate that acute sympathoexcitation constrains local vasodilation in the human skeletal muscle microvasculature during postocclusive reactive hyperemia. This finding indicates that integration of sympathetic vasoconstriction and local vasodilation is importantly involved in vascular regulation and the control of perfusion of the skeletal muscle microcirculation in humans.

Kir mediates Regenerative and Directional Conduction of Hyperpolarization in Brain Capillaries: Importance for Neurovascular Coupling

IntroductionRapid increase in local blood perfusion in response to elevated neuronal activity, a process referred to as neurovascular coupling (NVC), is crucial to the functioning and survival of neurons. Impairment of NVC is associated to a variety of neurological disorders including Alzheimer's disease, stroke, and vascular dementia [1]. Data by Longden et al. [2] suggest an active role of capillary endothelial cells (Cap‐ECs) in NVC. Inwardly rectifying potassium channels (Kir) enable capillaries to a) sense K+ release by nearby active neurons and b) amplify and conduct the resulting hyperpolarization to upstream feeding arterioles to increase cerebral blood flow (CBF) locally. Data by Twynstra et al. [3] in skeletal muscle microvasculature have also shown that propagating vasodilation becomes more specific in its direction as the branch order increases and that preferential conduction of vasodilatory signals in the upstream direction is possible through a yet unidentified mechanism. In this study, we use a mathematical model of a capillary network and its feeding parenchymal arteriole (PA), to examine the biophysical determinants that allow capillaries to sense modest elevations of K+ and transmit vasodilatory signals. We hypothesize that capillary level NVC is feasible through mechanisms that promote regenerative signal propagation in a preferentially upstream direction in the microvascular network.MethodsDetailed mathematical models of Cap‐ECs and PA ECs and smooth muscle cells (SMCs) are constructed. The models entail the dynamics of major ionic channels and intracellular components and predict membrane potential (Vm) and Ca2+ dynamics. Cap‐ECs are coupled through gap junctions to form a branched capillary network that is connected to a feeding PA (Fig 1A). Both local and distal hyperpolarizations are examined in response to K+ stimuli at different locations along the capillary network.ResultsSimulations predict a critical level of Kir density for generating local and conducted K+‐induced hyperpolarization in a series of connected Cap‐ECs (Fig 1B). When Kir current is small relative to the total transmembrane current, a passive spread of hyperpolarization is predicted (Fig. 1B; GKir,max ≈ 0.1 nS). Conversely, when Kir is dominant, the local hyperpolarization is significantly larger and the vasodilatory signal is conducted along the endothelial layer without signal attenuation (Fig 1B; GKir,max ≈ 0.3 nS). Furthermore, simulations in a simplified capillary network (Fig 1C), show that increasing Kir density upstream of bifurcations, allows preferential upstream conduction of the hyperpolarizing signal.ConclusionModel simulations suggest that Kir channels can mediate preferential and regenerative propagation of vasodilatory signals upstream the vascular network. This allows brain capillaries to effectively transmit vasodilatory signaling to feeding arterioles, in response to neuronal activity, and regulate local CBF.Support or Funding InformationThis work was supported by NIH grants R01HL131181 (to MTN), R01HL136636 (to FD), 1R15HL121778‐01A1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Clonogenic, myogenic progenitors expressing MCAM/CD146 are incorporated as adventitial reticular cells in the microvascular compartment of human post-natal skeletal muscle.

Recent observation identifies subendothelial (mural) cells expressing MCAM, a specific system of clonogenic, self-renewing, osteoprogenitors (a.k.a, “mesenchymal stem cells”) in the microvascular compartment of post-natal human bone marrow (BM). In this study, we used MCAM/CD146, as a marker to localize, isolate and assay subendothelial clonogenic cells from the microvasculature of postnatal human skeletal muscle. We show here that these cells share with their BM counterpart, anatomic position (subendothelial/adventitial) and ex vivo clonogenicity (CFU-Fs). When assayed under the stringent conditions, these cells display a high spontaneous myogenic potential (independent of co-culture with myoblasts or of in vivo fusion with local myoblasts), which is otherwise only attained in cultures of satellite cells. These muscle-derived mural cells activated a myogenic program in culture. Cultured CD146+ cells expressed the myogenic factors (Pax7, Pax3 and Myf5), NCAM/CD56, desmin as well as proteins characteristic of more advanced myogenic differentiation, such as myosin heavy chain. In vivo, these cells spontaneously generate myotubes and myofibrils. These data identify the anatomy and phenotype of a novel class of committed myogenic progenitor in human post-natal skeletal muscle of subendothelial cells associated with the abluminal surface of microvascular compartment distinct from satellite cells.

Diabetic Microvascular Disease: An Endocrine Society Scientific Statement.

Both type 1 and type 2 diabetes adversely affect the microvasculature in multiple organs. Our understanding of the genesis of this injury and of potential interventions to prevent, limit, or reverse injury/dysfunction is continuously evolving. This statement reviews biochemical/cellular pathways involved in facilitating and abrogating microvascular injury. The statement summarizes the types of injury/dysfunction that occur in the three classical diabetes microvascular target tissues, the eye, the kidney, and the peripheral nervous system; the statement also reviews information on the effects of diabetes and insulin resistance on the microvasculature of skin, brain, adipose tissue, and cardiac and skeletal muscle. Despite extensive and intensive research, it is disappointing that microvascular complications of diabetes continue to compromise the quantity and quality of life for patients with diabetes. Hopefully, by understanding and building on current research findings, we will discover new approaches for prevention and treatment that will be effective for future generations.

Enhancing Nitric Oxide Bioavailability via Exogen Nitric Oxide Synthase and L-Arginine Attenuates Ischemia-Reperfusion–Induced Microcirculatory Alterations

Nitric oxide (NO) is an important cytoprotective agent against ischemia and reperfusion injury (IRI). Enhancing NO bioavailability via exogen NO synthases (NOSs) and L-arginine promotes conversation to NO, circumventing the problem of nonfunctioning NOSs under hypoxic and acidic conditions. In this study, the authors evaluated the therapeutic efficacy of endothelial, inducible and neuronal NOS, and L-arginine on reperfusion-induced microcirculatory alterations and hemodynamic adverse effects in the microvasculature of skeletal muscle. Vascular pedicle isolated rat cremaster model was used that underwent 2 hours of warm ischemia followed by 1 hour of reperfusion. At 30 minutes before ischemia, normal saline (control group with/without ischemia), endothelial-, inducible-, and neuronal NOSs (2 IE) and L-arginine (50 mg/kg BW) were administered systemically (IV). Ischemia-reperfusion-induced microcirculatory alterations were measured after 1 hour of reperfusion. Mean arterial blood pressure and heart frequency were measured throughout the experiment to determine hemodynamic adverse effects. The isoforms of NOSs and L-arginine attenuated ischemia-reperfusion-induced vasoconstriction, improved red blood cell velocity, capillary flow, and leukocyte adherence to the endothelium wall. Hemodynamics was stable throughout the experiment. Enhancing NO bioavailability via exogen application of NOSs and L-arginine significantly attenuated ischemia-reperfusion-induced microcirculatory alterations in the microvasculature of skeletal muscle. Significant hemodynamic adverse effects were not present, thus demonstrating this approach might be useful for therapeutic intervention. This "pharmacologic preconditioning" could be an easy and effective interventional strategy to uphold conversation of L-arginine to NO under ischemic conditions.

Establishment of a new conditionally immortalized human skeletal muscle microvascular endothelial cell line.

In skeletal muscle, the capillaries have tight junctions (TJs) that are structurally similar to those in the blood-brain barrier (BBB) and blood-nerve barrier (BNB). Although many findings have been clarified in the territory of BBB and BNB, few have so far examined the TJs of capillaries in the skeletal muscle. In addition, no in vitro human skeletal muscle microvasculature models have been reported thus far. We newly established a new human skeletal muscle microvascular endothelial cell (HSMMEC) line. HSMMECs were isolated from human skeletal muscle and were infected with retroviruses harboring temperature-sensitive SV40 T antigen and telomerase genes. This cell line, termed TSM15, showed a spindle fiber-shaped morphology, an immunoreactivity to anti-factor VIII and anti-VE-cadherin antibodies, and a temperature-sensitive growth. TSM15 cells grew stably for more than 40 passages when they were cultured at 33°C, thereby retaining their spindle fiber-shaped morphology and contact inhibition at confluence. The cells expressed tight junctional molecules such as claudin-5, occludin, and zonula occludens-1, as well as transporters such as a glucose transporter 1. The transendothelial electrical resistance of TSM15 was as high as those of the human brain microvascular endothelial cell line. This novel cell line might facilitate the analyses of the pathophysiology of inflammatory myopathy, such as dermatomyositis, and can improve our understanding of the physiological and biochemical properties of the microvasculature in human skeletal muscle.

The effects of voluntary exercise and prazosin on capillary rarefaction and metabolism in streptozotocin-induced diabetic male rats.

Type-1 diabetes mellitus (T1D) causes impairments within the skeletal muscle microvasculature. Both regular exercise and prazosin have been shown to improve skeletal muscle capillarization and metabolism in healthy rats through distinct angiogenic mechanisms. The aim of this study was to evaluate the independent and additive effects of voluntary exercise and prazosin treatment on capillary-to-fiber ratio (C:F) in streptozotocin (STZ)-treated diabetic rats. STZ (65 mg/kg) was intraperitoneally administered to male Sprague-Dawley rats (n = 36) to induce diabetes, with healthy, nondiabetic, sedentary rats (n = 10) as controls. The STZ-treated rats were then divided into sedentary (SED) or exercising (EX; 24-h access to running wheels) groups and then further subdivided into prazosin (Praz) or water (H2O) treatment groups: nondiabetic-SED-H2O, STZ-SED-H2O, STZ-EX-H2O, STZ-SED-Praz, and STZ-EX-Praz. After 3 wk, untreated diabetes significantly reduced the C:F in tibialis anterior (TA) and soleus muscles in the STZ-SED-H2O animals (both P < 0.05). Voluntary exercise and prazosin treatment independently resulted in a normalization of C:F within the TA (1.86 ± 0.12 and 2.04 ± 0.03 vs 1.71 ± 0.09, P < 0.05) and the soleus (2.36 ± 0.07 and 2.68 ± 0.14 vs 2.13 ± 0.12, P < 0.05). The combined STZ-EX-Praz group resulted in the highest C:F within the TA (2.26 ± 0.07, P < 0.05). Voluntary exercise volume was negatively correlated with fed blood glucose levels (r2 = -0.7015, P < 0.01) and, when combined with prazosin, caused further enhanced nonfasted glucose (P < 0.01). Exercise and prazosin reduced circulating nonesterified fatty acids more than either stimulus alone (P < 0.05). These results suggest that the distinct stimulation of angiogenesis, with both regular exercise and prazosin treatment, causes a cooperative improvement in the microvascular complications associated with T1D.NEW & NOTEWORTHY It is currently well established that poorly controlled diabetes reduces both skeletal muscle mass and muscle capillarization. These muscle-specific features of diabetes may, in turn, compromise insulin sensitivity and glucose control. Using a model of streptozotocin-induced diabetes, we show the vascular complications linked with disease and how chronic exposure to exercise and prazosin (an α1-adrenergic antagonist) can reduce these complications and improve glycemic control.

Immunomodulatory capacity of the local mesenchymal stem cells transplantation after severe skeletal muscle injury in female rats

Context: Cell therapy technique with stem cells is a very attractive strategy for the treatment of muscle disorders.Objective: The objective of this study was to investigate the mechanism of local transplantation of mesenchymal stem cells (MSCs) which could contribute to skeletal muscle healing.Materials and methods: Female rats were divided into three equal groups as the following: group 1, the negative control group (untreated group), group 2, sham-treated group, rats with muscle injuries involving volumetric muscle loss (VML) of adductor brevis muscle and injected locally with phosphate-buffered saline (PBS) 0.5 ml without stem cells after 7 d of muscle injury, group 3, treated group, rats with VML and injected locally (intramuscular) with 1.5 × 106 bone marrow MSCs suspended in PBS 0.5 ml (1) after 7 d of muscle tissue injury. All animals were sacrificed after 4 weeks of stem cell transplantation.Results: In vitro culture the morphology of MSCs reached confluence and appeared as long spindle in shape on 9–14 d. Most of the cells did not express the hematopoietic cell marker, CD34 and CD45 but expressed MSCs marker CD44, CD90 and CD105. The remarkable increase of proliferating cell nuclear antigen positive nucleus was recorded in MSCs group as compared to PBS group. After 28 d of injection, administration of only PBS into the site of muscle injury caused up-regulation in the levels of interleukins IL-1β, IL-6, tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β1), interferon alpha (IFN-α) and down-regulate the level of IL-10 in muscular tissue comparing to the untreated control. Bone marrow MSCs + PBS injected at the site of muscle injury significantly down-regulate the inflammatory cytokines levels IL-1β and IL-6 and TNF-α, TGF-β1 and IFN-α and up-regulate the level of IL-10. Collagen concentrations in the injured skeletal muscle estimated by enzyme-linked immuno sorbent assay and stained with Masson trichrome stain were increased with PBS group and decreased after transplantation of bone marrow MSCs in the site of injury. Muscle sections stained with H&E showed a higher number of centronucleated regenerating myofibers in the stem-cell-treated group than in the (PBS) and untreated control group. Microvasculature of skeletal muscle was decreased as demonstrated by immunostaining technique for CD34 in PBS group from untreated control. The MSCs group showed angiogenesis and marked increase of skeletal muscle microvasculature than PBS group.Conclusion: MSCs can modify the local immunological responses and improve muscle regeneration by suppressing of inflammatory cytokines, activating of the anti-inflammatory cytokine, restoration of muscle fibers and angiogenesis. By means of increase in TGF-β production in response to muscle injury prevent the repair of injured fibers and increase connective tissue production (collagen fibers), thus propagating skeletal muscle weakness and fibrosis whereas MSCs + PBS injected at the site of muscle injury significantly down-regulate (TGF-β1) and hence the level of collagen (fibrosis or scar areas). MSCs are able to block the fibrotic signaling cascade by declining TGF-β1 and scar areas in the injured muscle.

Tissue Inhibitor of Metalloproteinase 1 Influences Vascular Adaptations to Chronic Alterations in Blood Flow.

Remodeling of the skeletal muscle microvasculature involves the coordinated actions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs). We hypothesized that the loss of TIMP1 would enhance both ischemia and flow-induced vascular remodeling by increasing MMP activity. TIMP1 deficient (Timp1-/- ) and wild-type (WT) C57BL/6 mice underwent unilateral femoral artery (FA) ligation or were treated with prazosin, an alpha-1 adrenergic receptor antagonist, in order to investigate vascular remodeling to altered flow. Under basal conditions, Timp1-/- mice had reduced microvascular content as compared to WT mice. Furthermore, vascular remodeling was impaired in Timp1-/- mice. Timp1-/- mice displayed reduced blood flow recovery in response to FA ligation and no arteriogenic response to prazosin treatment. Timp1-/- mice failed to undergo angiogenesis in response to ischemia or prazosin, despite maintaining the capacity to increase VEGF-A and eNOS mRNA. Vascular permeability was increased in muscles of Timp1-/- mice in response to both prazosin treatment and FA ligation, but this was not accompanied by greater MMP activity. This study highlights a previously undescribed integral role for TIMP1 in both vascular network maturation and adaptations to ischemia or alterations in flow. J. Cell. Physiol. 232: 831-841, 2017. © 2016 Wiley Periodicals, Inc.

Role of Growth Factors in Modulation of the Microvasculature in Adult Skeletal Muscle.

Post-natal skeletal muscle is a highly plastic tissue that has the capacity to regenerate rapidly following injury, and to undergo significant modification in tissue mass (i.e. atrophy/hypertrophy) in response to global metabolic changes. These processes are reliant largely on soluble factors that directly modulate muscle regeneration and mass. However, skeletal muscle function also depends on an adequate blood supply. Thus muscle regeneration and changes in muscle mass, particularly hypertrophy, also demand rapid changes in the microvasculature. Recent evidence clearly demonstrates a critical role for soluble growth factors in the tight regulation of angiogenic expansion of the muscle microvasculature. Furthermore, exogenous modulation of these factors has the capacity to impact directly on angiogenesis and thus, indirectly, on muscle regeneration, growth and performance. This chapter reviews recent developments in understanding the role of growth factors in modulating the skeletal muscle microvasculature, and the potential therapeutic applications of exogenous angiogenic and anti-angiogenic mediators in promoting effective growth and regeneration, and ameliorating certain diseases, of skeletal muscle.