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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Differential changes in blood flow and oxygen utilization in active muscles between voluntary exercise and electrical muscle stimulation in young adults.

The physiological effects on blood flow and oxygen utilization in active muscles during and after involuntary contraction triggered by electrical muscle stimulation (EMS) remain unclear, particularly compared with those elicited by voluntary (VOL) contractions. Therefore, we used diffuse correlation and near-infrared spectroscopy (DCS-NIRS) to compare changes in local muscle blood flow and oxygen consumption during and after these two types of muscle contractions in humans. Overall, 24 healthy young adults participated in the study, and data were successfully obtained from 17 of them. Intermittent (2-s contraction, 2-s relaxation) isometric ankle dorsiflexion with a target tension of 20% of maximal VOL contraction was performed by EMS or VOL for 2 min, followed by a 6-min recovery period. DCS-NIRS probes were placed on the tibialis anterior muscle, and relative changes in local tissue blood flow index (rBFI), oxygen extraction fraction (rOEF), and metabolic rate of oxygen (rMRO2) were continuously derived. EMS induced more significant increases in rOEF and rMRO2 than VOL exercise but a comparable increase in rBFI. After EMS, rBFI and rMRO2 decreased more slowly than after VOL and remained significantly higher until the end of the recovery period. We concluded that EMS augments oxygen consumption in contracting muscles by enhancing oxygen extraction while increasing oxygen delivery at a rate similar to the VOL exercise. Under the conditions examined in this study, EMS demonstrated a more pronounced and/or prolonged enhancement in local muscle perfusion and aerobic metabolism compared with VOL exercise in healthy participants.NEW & NOTEWORTHY This is the first study to visualize continuous changes in blood flow and oxygen utilization within contracted muscles during and after electrical muscle stimulation (EMS) using combined diffuse correlation and near-infrared spectroscopy. We found that initiating EMS increases blood flow at a rate comparable to that during voluntary (VOL) exercise but enhances oxygen extraction, resulting in higher oxygen consumption. Furthermore, EMS increased postexercise muscle perfusion and oxygen consumption compared with that after VOL exercise.

An exercise stress test for contrast-enhanced duplex ultrasound assessment of lower limb muscle perfusion in patients with peripheral arterial disease.

OBJECTIVE: The aim of the present study was to develop a standardized contrast-enhanced duplex ultrasound (CE-DUS) protocol to assess lower-extremity muscle perfusion before and after exercise and determine relationships of perfusion with clinical and functional measures. METHODS: CE-DUS (EPIQ 5G, Philips) was used before and immediately after a 10-minute, standardized bout of treadmill walking to compare microvascular perfusion of the gastrocnemius muscle in older (55-82 years) patients with peripheral arterial disease (PAD) (n = 15, mean ankle-brachial index, 0.78 ± 0.04) and controls (n = 13). Microvascular blood volume (MBV) and microvascular flow velocity (MFV) were measured at rest and immediately following treadmill exercise, and the Modified Physical Performance Test (MPPT) was used to assess mobility function. RESULTS: In the resting state (pre-exercise), MBV in patients with PAD was not significantly different than normal controls (5.17 ± 0.71 vs 6.20 ± 0.83 arbitrary units (AU) respectively; P = .36); however, after exercise, MBV was ∼40% lower in patients with PAD compared with normal controls (5.85 ± 1.13 vs 9.53 ± 1.31 AU, respectively; P = .04). Conversely, MFV was ∼60% higher in patients with PAD compared with normal controls after exercise (0.180 ± 0.016 vs 0.113 ± 0.018 AU, respectively; P = .01). There was a significant between-group difference in the exercise-induced changes in both MBV and MFV (P ≤ .05). Both basal and exercise MBV directly correlated with MPPT score in the patients with PAD (r = 0.56-0.62; P < .05). CONCLUSIONS: This standardized protocol for exercise stress testing of the lower extremities quantifies calf muscle perfusion and elicits perfusion deficits in patients with PAD. This technique objectively quantifies microvascular perfusion deficits that are related to reduced mobility function and could be used to assess therapeutic efficacy in patients with PAD.

A machine learning-based approach to identify peripheral artery disease using texture features from contrast-enhanced magnetic resonance imaging.

Diagnosing and assessing the risk of peripheral artery disease (PAD) has long been a focal point for medical practitioners. The impaired blood circulation in PAD patients results in altered microvascular perfusion patterns in the calf muscles which is the primary location of intermittent claudication pain. Consequently, we hypothesized that changes in perfusion and increase in connective tissue could lead to alterations in the appearance or texture patterns of the skeletal calf muscles, as visualized with non-invasive imaging techniques. We designed an automatic pipeline for textural feature extraction from contrast-enhanced magnetic resonance imaging (CE-MRI) scans and used the texture features to train machine learning models to detect the heterogeneity in the muscle pattern among PAD patients and matched controls. CE-MRIs from 36 PAD patients and 20 matched controls were used for preparing training and testing data at a 7:3 ratio with cross-validation (CV) techniques. We employed feature arrangement and selection methods to optimize the number of features. The proposed method achieved a peak accuracy of 94.11% and a mean testing accuracy of 84.85% in a 2-class classification approach (controls vs. PAD). A three-class classification approach was performed to identify a high-risk PAD sub-group which yielded an average test accuracy of 83.23% (matched controls vs. PAD without diabetes vs. PAD with diabetes). Similarly, we obtained 78.60% average accuracy among matched controls, PAD treadmill exercise completers, and PAD exercise treadmill non-completers. Machine learning and imaging-based texture features may be of interest in the study of lower extremity ischemia.

Pedicled Peroneus Brevis Muscle Flaps as an Alternative to Fasciocutaneous Rotational Flaps for Lower-Extremity Soft Tissue Defects.

OBJECTIVES: To report our experience using a peroneus brevis flap (PBF) for soft tissue defects of the distal third of the tibia, ankle, and hindfoot in resource-challenged environments. DESIGN: Retrospective review. SETTING: Rural outpatient surgical facility in Honduras. PATIENT SELECTION CRITERIA: Patients who sustained tibia, ankle, or hindfoot fractures or traumatic degloving, with critical-sized soft tissue defects treated with either a proximally based or distally based pedicled PBF to achieve coverage of the middle and distal third of the leg, ankle, and/or hindfoot. OUTCOME MEASURES AND COMPARISONS: Flap healing, complications, and reoperations. RESULTS: Twenty-three patients, 4 with proximally based and 19 with distally based PBF flaps were included. The mean patient age was 37.3 (SD = 18.3; range 18-75 years). Duration of follow-up averaged 14.7 months (SD = 11.4; range 4-46). The PBF successfully covered the defect without the need for additional unplanned surgical flap coverage in all but 2 patients. Thirty percent of the PBFs received a split thickness skin graft, while the remainder granulated successfully without skin graft. Four flaps were partially debrided without additional flap mobilization, while 1 flap was lost completely. Ten patients had successful re-elevation of their flaps for secondary procedures such as implant removal, spacer exchange, deep debridements, and bone grafting. All donor site incisions healed without complication. CONCLUSIONS: The pedicled PBF allows coverage of distal leg, ankle, and hindfoot wounds using muscle in patients who may otherwise require free tissue flaps or transfer to another institution for coverage. PBFs can be learned and implemented without the use of microvascular techniques. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Co-Delivery of Bioengineered Exosomes and Oxygen for Treating Critical Limb Ischemia in Diabetic Mice.

Diabetic patients with critical limb ischemia face a high rate of limb amputation. Regeneration of the vasculature and skeletal muscles can salvage diseased limbs. Therapy using stem cell-derived exosomes that contain multiple proangiogenic and promyogenic factors represents a promising strategy. Yet the therapeutic efficacy is not optimal because exosomes alone cannot efficiently rescue and recruit endothelial and skeletal muscle cells and restore their functions under hyperglycemic and ischemic conditions. To address these limitations, we fabricated ischemic-limb-targeting stem cell-derived exosomes and oxygen-releasing nanoparticles and codelivered them in order to recruit endothelial and skeletal muscle cells, improve cell survival under ischemia before vasculature is established, and restore cell morphogenic function under high glucose and ischemic conditions. The exosomes and oxygen-releasing nanoparticles, delivered by intravenous injection, specifically accumulated in the ischemic limbs. Following 4 weeks of delivery, the exosomes and released oxygen synergistically stimulated angiogenesis and muscle regeneration without inducing substantial inflammation and reactive oxygen species overproduction. Our work demonstrates that codelivery of exosomes and oxygen is a promising treatment solution for saving diabetic ischemic limbs.

Exercise and Ischemia-Activated Pathways in Limb Muscle Angiogenesis and Vascular Regeneration.

Exercise has a profound effect on cardiovascular disease, particularly through vascular remodeling and regeneration. Peripheral artery disease (PAD) is one such cardiovascular condition that benefits from regular exercise or rehabilitative physical therapy in terms of slowing the progression of disease and delaying amputations. Various rodent pre-clinical studies using models of PAD and exercise have shed light on molecular pathways of vascular regeneration. Here, I review key exercise-activated signaling pathways (nuclear receptors, kinases, and hypoxia inducible factors) in the skeletal muscle that drive paracrine regenerative angiogenesis. The rationale for highlighting the skeletal muscle is that it is the largest organ recruited during exercise. During exercise, skeletal muscle releases several myokines, including angiogenic factors and cytokines that drive tissue vascular regeneration via activation of endothelial cells, as well as by recruiting immune and endothelial progenitor cells. Some of these core exercise-activated pathways can be extrapolated to vascular regeneration in other organs. I also highlight future areas of exercise research (including metabolomics, single cell transcriptomics, and extracellular vesicle biology) to advance our understanding of how exercise induces vascular regeneration at the molecular level, and propose the idea of "exercise-mimicking" therapeutics for vascular recovery.

Regulation of Skeletal Muscle Resistance Arteriolar Tone: Integration of Multiple Mechanisms.

INTRODUCTION: Physiological system complexity represents an imposing challenge to gaining insight into how arteriolar behavior emerges. Further, mechanistic complexity in arteriolar tone regulation requires that a systematic determination of how these processes interact to alter vascular diameter be undertaken. METHODS: The present study evaluated the reactivity of ex vivo proximal and in situ distal resistance arterioles in skeletal muscle with challenges across the full range of multiple physiologically relevant stimuli and determined the stability of responses over progressive alterations to each other parameter. The five parameters chosen for examination were (1) metabolism (adenosine concentration), (2) adrenergic activation (norepinephrine concentration), (3) myogenic activation (intravascular pressure), (4) oxygen (superfusate PO2), and (5) wall shear rate (altered intraluminal flow). Vasomotor tone of both arteriole groups following challenge with individual parameters was determined; subsequently, responses were determined following all two- and three-parameter combinations to gain deeper insight into how stimuli integrate to change arteriolar tone. A hierarchical ranking of stimulus significance for establishing arteriolar tone was performed using mathematical and statistical analyses in conjunction with machine learning methods. RESULTS: Results were consistent across methods and indicated that metabolic and adrenergic influences were most robust and stable across all conditions. While the other parameters individually impact arteriolar tone, their impact can be readily overridden by the two dominant contributors. CONCLUSION: These data suggest that mechanisms regulating arteriolar tone are strongly affected by acute changes to the local environment and that ongoing investigation into how microvessels integrate stimuli regulating tone will provide a more thorough understanding of arteriolar behavior emergence across physiological and pathological states.

Venous congestion affects neuromuscular changes in pigs in terms of muscle electrical activity and muscle stiffness.

Early detection of venous congestion (VC)-related diseases such as deep vein thrombosis (DVT) is important to prevent irreversible or serious pathological conditions. However, the current way of diagnosing DVT is only possible after recognizing advanced DVT symptoms such as swelling, pain, and tightness in affected extremities, which may be due to the lack of information on neuromechanical changes following VC. Thus, the goal of this study was to investigate acute neuromechanical changes in muscle electrical activity and muscle stiffness when VC was induced. The eight pigs were selected and the change of muscle stiffness from the acceleration and muscle activity in terms of integral electromyography (IEMG) was investigated in three VC stages. Consequently, we discovered a significant increase in the change in muscle stiffness and IEMG from the baseline to the VC stages (p < 0.05). Our results and approach can enable early detection of pathological conditions associated with VC, which can be a basis for further developing early diagnostic tools for detecting VC-related diseases.

Estrogen-Related Receptor Gamma Gene Therapy Promotes Therapeutic Angiogenesis and Muscle Recovery in Preclinical Model of PAD.

Background Peripheral arterial disease and critical limb ischemia are cardiovascular complications associated with vascular insufficiency, oxidative metabolic dysfunction, and myopathy in the limbs. Estrogen-related receptor gamma (ERRγ) has emerged as a dual regulator of paracrine angiogenesis and oxidative metabolism through transgenic mouse studies. Here our objective was to investigate whether postischemic intramuscular targeting of ERRγ via gene therapy promotes ischemic recovery in a preclinical model of peripheral arterial disease/critical limb ischemia. Methods and Results Adeno-associated virus 9 (AAV9) Esrrg gene delivery vector was developed and first tested via intramuscular injection in murine skeletal muscle. AAV9-Esrrg robustly increased ERRγ protein expression, induced angiogenic and oxidative genes, and boosted capillary density and succinate dehydrogenase oxidative metabolic activity in skeletal muscles of C57Bl/6J mice. Next, hindlimb ischemia was induced via unilateral femoral vessel ligation in mice, followed by intramuscular AAV9-Esrrg (or AAV9-green fluorescent protein) gene delivery 24 hours after injury. ERRγ overexpression increased ischemic neoangiogenesis and markers of endothelial activation, and significantly improved ischemic revascularization measured using laser Doppler flowmetry. Moreover, ERRγ overexpression restored succinate dehydrogenase oxidative metabolic capacity in ischemic muscle, which correlated with increased mitochondrial respiratory complex protein expression. Most importantly, myofiber size to number quantification revealed that AAV9-Esrrg restores myofibrillar size and mitigates ischemia-induced myopathy. Conclusions These results demonstrate that intramuscular AAV9-Esrrg delivery rescues ischemic pathology after hindlimb ischemia, underscoring that Esrrg gene therapy or pharmacological activation could be a promising strategy for the management of peripheral arterial disease/critical limb ischemia.

Evidence of impaired functional sympatholysis in patients with heart failure with preserved ejection fraction.

Exercising muscle blood flow is reduced in patients with heart failure with a preserved ejection fraction (HFpEF), which may be related to disease-related changes in the ability to overcome sympathetic nervous system (SNS)-mediated vasoconstriction during exercise, (i.e., "functional sympatholysis"). Thus, in 12 patients with HFpEF (69 ± 7 yr) and 11 healthy controls (Con, 69 ± 4 yr), we examined forearm blood flow (FBF), mean arterial pressure (MAP), and forearm vascular conductance (FVC) during rhythmic handgrip exercise (HG) at 30% of maximum voluntary contraction with or without lower-body negative pressure (LBNP, -20 mmHg) to increase SNS activity and elicit peripheral vasoconstriction. SNS-mediated vasoconstrictor responses were determined as LBNP-induced changes (%Δ) in FVC, and the "magnitude of sympatholysis" was calculated as the difference between responses at rest and during exercise. At rest, the LBNP-induced change in FVC was significantly lesser in HFpEF compared with Con (HFpEF: -9.5 ± 5.5 vs. Con: -21.0 ± 8.0%; P < 0.01). During exercise, LBNP-induced %ΔFVC was significantly attenuated in Con compared with rest (HG: -5.8 ± 6.0%; P < 0.05) but not in HFpEF (HG: -9.9 ± 2.5%; P = 0.88). Thus, the magnitude of sympatholysis was lesser in HFpEF compared with Con (HFpEF: 0.4 ± 4.7 vs. Con: -15.2 ± 11.8%; P < 0.01). These data demonstrate a diminished ability to attenuate SNS-mediated vasoconstriction in HFpEF and provide new evidence suggesting impaired functional sympatholysis in this patient group.NEW & NOTEWORTHY Data from the current study suggest that functional sympatholysis, or the ability to adequately attenuate sympathetic nervous system (SNS)-mediated vasoconstriction during exercise, is impaired in patients with heart failure with preserved ejection fraction (HFpEF). These observations extend the current understanding of HFpEF pathophysiology by implicating inadequate functional sympatholysis as an important contributor to reduced exercising muscle blood flow in this patient group.

A single high-fat Western meal modulates vascular responsiveness to sympathetic activation at rest and during exercise in humans: a randomized controlled trial.

A single high-fat Western meal transiently reduces endothelium-dependent vasodilation at rest, but the interaction with sympathetic vasoconstrictor activity during exercise remains unknown. Herein, we tested the hypothesis that a single high-fat Western meal would impair the ability of contracting skeletal muscle to offset vascular responsiveness to sympathetic activation during exercise, termed functional sympatholysis. In 18 (10 females/8 males) healthy young adults, forearm blood flow (Doppler ultrasound) and beat-to-beat arterial pressure (photoplethysmography) were measured during lower-body negative pressure (LBNP; -20 mmHg) applied at rest and simultaneously during low (15% maximum contraction) and moderate (30% maximum contraction)-intensity rhythmic handgrip exercise. The magnitude of sympatholysis was calculated as the difference of LBNP-induced changes in forearm vascular conductance (FVC) between handgrip and rest. Experiments were performed preprandial and 1 h, 2 h, and 3 h after a high- or low-fat meal. In the preprandial state, LBNP decreased resting FVC (Δ-54 ± 10%), and these responses were attenuated during low (Δ-17 ± 7%)- and moderate (Δ-8 ± 6%)-intensity handgrip exercise. Following a high-fat meal, LBNP induced attenuated decreases in resting FVC (3 h postprandial, Δ-47 ± 10%, P = 0.002 vs. preprandial) and blunted attenuation of FVC during low (3 h postprandial, Δ-23 ± 8%, P = 0.001 vs. preprandial)- and moderate (3 h postprandial, Δ-16 ± 6%, P < 0.001 vs. preprandial)-intensity handgrip exercise. The high-fat meal attenuated the magnitude of sympatholysis during low (preprandial, 38 ± 7 vs. 3 h postprandial, 23 ± 8%, P < 0.001)- and moderate (preprandial, 46 ± 11 vs. 3 h postprandial, 31 ± 10%, P < 0.001)-intensity handgrip exercise. The low-fat meal had no impact on these responses. In conclusion, a single high-fat Western meal modulates sympathetic vasoconstriction at rest and during low- and moderate-intensity handgrip exercise in young healthy adults.NEW & NOTEWORTHY We observed that a single high-fat Western meal, but not an isocaloric low-fat meal, attenuated the sympathetic vasoconstriction at rest and the ability of the active skeletal muscle to counteract the vascular responsiveness to sympathetic activation (i.e., functional sympatholysis) during low- and moderate-intensity rhythmic handgrip exercise in healthy young adults. Our findings highlight the potential deleterious vascular effect associated with the consumption of a Western diet.

Autonomic cardiovascular control during exercise.

The cardiovascular response to exercise is largely determined by neurocirculatory control mechanisms that help to raise blood pressure and modulate vascular resistance which, in concert with regional vasodilatory mechanisms, promote blood flow to active muscle and organs. These neurocirculatory control mechanisms include a feedforward mechanism, known as central command, and three feedback mechanisms, namely, 1) the baroreflex, 2) the exercise pressor reflex, and 3) the arterial chemoreflex. The hemodynamic consequences of these control mechanisms result from their influence on the autonomic nervous system and subsequent alterations in cardiac output and vascular resistance. Although stimulation of the baroreflex inhibits sympathetic outflow and facilitates parasympathetic activity, central command, the exercise pressor reflex, and the arterial chemoreflex facilitate sympathetic activation and inhibit parasympathetic drive. Despite considerable understanding of the cardiovascular consequences of each of these mechanisms in isolation, the circulatory impact of their interaction, which occurs when various control systems are simultaneously activated (e.g., during exercise at altitude), has only recently been recognized. Although aging and cardiovascular disease (e.g., heart failure, hypertension) have both been recognized to alter the hemodynamic consequences of these regulatory systems, this review is limited to provide a brief overview on the action and interaction of neurocirculatory control mechanisms in health.

Redefining the vascular anatomy of the medial gastrocnemius muscle: A computed tomography angiography study.

BACKGROUND: The medial gastrocnemius (GN) muscle flap is a historical reconstructive option in lower limb reconstruction. The flap is proximally based on the medial sural artery, and it is assumed not possible to harvest a distally based flap because of the absence of other minor pedicles. The aim of this study is to investigate the presence and the anatomy of a distal secondary pedicle given off by the posterior tibial artery (PTA). METHODS: A retrospective CTA study was performed of 120 limbs between April 2018 and June 2020. 3D reconstruction was performed to delineate the anatomy of the distal secondary pedicle, if present. The distance of the pedicle, if found, from the intermalleolar line to the patella was noted. The number of pedicles, if multiple, was documented, as well as branches to the soleus muscle and the skin. RESULTS: A distal pedicle to the gastrocnemius muscle was found in 64% of limbs. The average location from the intermalleolar line is 168 mm. The branching pattern from the PTA showed an isolated vessel going to the distal medial gastrocnemius (32.8%), two branches to the medial gastrocnemius and skin (39.3%), two branches to the medial gastrocnemius and soleus (24.6%), and three branches to the medial gastrocnemius, soleus, and the skin (3.3%). CONCLUSIONS: This study confirms the presence of the secondary axial distal pedicle of the GN muscle. Furthermore, this study confirms that there is a likely association between the distal medial gastrocnemius pedicle and the PTA skin perforators.

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.

Reactive hyperemia half-time response is associated with skeletal muscle oxygen saturation changes during cycling exercise.

We investigated the relationship between muscle microvascular responses during reactive hyperemia as assessed using near-infrared spectroscopy (NIRS) with changes in skeletal muscle oxygen saturation during exercise. Thirty young untrained adults (M/W: 20/10; 23 ± 5 years) completed a maximal cycling exercise test to determine exercise intensities performed on a subsequent visit separated by seven days. At the second visit, post-occlusive reactive hyperemia was measured as changes in NIRS-derived tissue saturation index (TSI) at the left vastus lateralis muscle. Variables of interest included desaturation magnitude, resaturation rate, resaturation half-time, and hyperemic area under the curve. Afterwards, two 4-minute bouts of moderate intensity cycling followed by one bout of severe intensity cycling to fatigue took place while TSI was measured at the vastus lateralis muscle. TSI was averaged across the last 60-s of each moderate intensity bout then averaged together for analysis, and at 60-s into severe exercise. The change in TSI (∆TSI) during exercise is expressed relative to a 20 W cycling baseline. On average, the ΔTSI was -3.4 ± 2.4 % and -7.2 ± 2.8 % during moderate and severe intensity cycling, respectively. Resaturation half-time was correlated with the ΔTSI during moderate (r = -0.42, P = 0.01) and severe (r = -0.53, P = 0.002) intensity exercise. No other reactive hyperemia variable was found to correlate with ΔTSI. These results indicate that resaturation half-time during reactive hyperemia represents a resting muscle microvascular measure that associates with the degree of skeletal muscle desaturation during exercise in young adults.

Capillary-Mitochondrial Oxygen Transport in Muscle: Paradigm Shifts.

When exercising humans increase their oxygen uptake (V̇O2) 20-fold above rest the numbers are staggering: Each minute the O2 transport system - lungs, cardiovascular, active muscles - transports and utilizes 161 sextillion (10 21) O2 molecules. Leg extension exercise increases the quadriceps muscles' blood flow 100-times; transporting 17 sextillion O2 molecules per kilogram per minute from microcirculation (capillaries) to mitochondria powering their cellular energetics. Within these muscles, the capillary network constitutes a prodigious blood-tissue interface essential to exchange O2 and carbon dioxide requisite for muscle function. In disease, microcirculatory dysfunction underlies the pathophysiology of heart failure, diabetes, hypertension, pulmonary disease, sepsis, stroke and senile dementia. Effective therapeutic countermeasure design demands knowledge of microvascular/capillary function in health to recognize and combat pathological dysfunction. Dated concepts of skeletal muscle capillary (from the Latin capillus meaning 'hair') function prevail despite rigorous data-supported contemporary models; hindering progress in the field for future and current students, researchers and clinicians. Following closely the 100th anniversary of August Krogh's 1920 Nobel Prize for capillary function this Evidence Review presents an anatomical and physiological development of this dynamic field: Constructing a scientifically defensible platform for our current understanding of microcirculatory physiological function in supporting blood-mitochondrial O2 transport. New developments include: 1. Putative roles of red blood cell aquaporin and rhesus channels in determining tissue O2 diffusion. 2. Recent discoveries regarding intramyocyte O2 transport. 3. Developing a comprehensive capillary functional model for muscle O2 delivery-to-V̇O2 matching. 4. Use of kinetics analysis to discriminate control mechanisms from collateral or pathological phenomena.

5-HT7 receptors mediate dilation of rat cremaster muscle arterioles in vivo.

OBJECTIVE: Serotonin (5-HT) infusion in vivo causes hypotension and a fall in total peripheral resistance. However, the vascular segment and the receptors that mediate this response remain in question. We hypothesized that 5-HT7 receptors mediate arteriolar dilation to 5-HT in skeletal muscle microcirculation. METHODS: Cremaster muscles of isoflurane-anesthetized male Sprague-Dawley rats were prepared for in vivo microscopy of third- and fourth-order arterioles and superfused with physiological salt solution at 34°C. Quantitative real-time PCR (RT-PCR) was applied to pooled samples of first- to third-order cremaster arterioles (2-4 rats/sample) to evaluate 5-HT7 receptor expression. RESULTS: Topical 5-HT (1-10 nmols) or the 5-HT1/7 receptor agonist, 5-carboxamidotryptamine (10-30 nM), dilated third- and fourth-order arterioles, responses that were abolished by 1 µM SB269970, a selective 5-HT7 receptor antagonist. In contrast, dilation induced by the muscarinic agonist, methacholine (100 nmols) was not inhibited by SB269970. Serotonin (10 nmols) failed to dilate cremaster arterioles in 5-HT7 receptor knockout rats whereas arterioles in wild-type litter mates dilated to 1 nmol 5-HT, a response blocked by 1 µM SB269970. Quantitative RT-PCR revealed that cremaster arterioles expressed mRNA for 5-HT7 receptors. CONCLUSIONS: 5-HT7 receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to 5-HT-induced hypotension, in vivo.

E-Selectin/AAV Gene Therapy Promotes Myogenesis and Skeletal Muscle Recovery in a Mouse Hindlimb Ischemia Model.

The response to ischemia in peripheral artery disease (PAD) depends on compensatory neovascularization and coordination of tissue regeneration. Identifying novel mechanisms regulating these processes is critical to the development of nonsurgical treatments for PAD. E-selectin is an adhesion molecule that mediates cell recruitment during neovascularization. Therapeutic priming of ischemic limb tissues with intramuscular E-selectin gene therapy promotes angiogenesis and reduces tissue loss in a murine hindlimb gangrene model. In this study, we evaluated the effects of E-selectin gene therapy on skeletal muscle recovery, specifically focusing on exercise performance and myofiber regeneration. C57BL/6J mice were treated with intramuscular E-selectin/adeno-associated virus serotype 2/2 gene therapy (E-sel/AAV) or LacZ/AAV2/2 (LacZ/AAV) as control and then subjected to femoral artery coagulation. Recovery of hindlimb perfusion was assessed by laser Doppler perfusion imaging and muscle function by treadmill exhaustion and grip strength testing. After three postoperative weeks, hindlimb muscle was harvested for immunofluorescence analysis. At all postoperative time points, mice treated with E-sel/AAV had improved hindlimb perfusion and exercise capacity. E-sel/AAV gene therapy also increased the coexpression of MyoD and Ki-67 in skeletal muscle progenitors and the proportion of Myh7+ myofibers. Altogether, our findings demonstrate that in addition to improving reperfusion, intramuscular E-sel/AAV gene therapy enhances the regeneration of ischemic skeletal muscle with a corresponding benefit on exercise performance. These results suggest a potential role for E-sel/AAV gene therapy as a nonsurgical adjunct in patients with life-limiting PAD.