Hindlimb Blood Flow Research Articles

1936-P: Muscle Ischemia Impairs Insulin-Mediated Microvascular Recruitment

Diabetes predisposes patients to peripheral artery disease (PAD). Muscle microvasculature plays a critical role in maintaining muscle function and health by delivering oxygen, nutrients and hormones to myocytes. Insulin dilates precapillary arterioles to increase muscle perfusion. To define the impact of ischemia to muscle microvascular perfusion and microvascular response to insulin, adult male rats were subject to hind limb ischemic surgery. Total hind limb blood flow was determined using laser Doppler blood flowmetry and muscle microvascular blood volume (MBV) measured using contrast-enhanced ultrasound in both the ischemic (left) and control (right) hind limb. Ischemic to control limb (I/C) ratios were calculated. Hyperinsulinemic euglycemia clamps were performed to determine microvascular responses to insulin. Hind limb ischemic surgery decreased total limb blood flow by ∼70% immediately, from a pre-surgical I/C ratio of 1.10 ± 0.07 to 0.34 ± 0.06. The I/C ratios then progressively increased to 0.45 ± 0.08, 0.54 ± 0.06, 0.67 ± 0.11, and 0.74± 0.11 on postsurgical day 3, 7, 14 and 21 respectively. Similarly, ischemic surgery decreased MBV promptly from a pre-surgical I/C ratio of 1.01 ± 0.04 to 0.39 ± 0.06. MBV I/C ratios completely recovered on day 3 (1.11 ± 0.09) and remained stable afterwards. Insulin induced similar increases in muscle MBV in both legs before surgery (I/C ratio 1.14 ± 0.16) but the increase was much less in ischemic leg on day 3 (I/C ratio 0.63 ± 0.15). Microvascular insulin responses fully recovered on day 7 (I/C ratio 0.97± 0.25). We conclude that hind limb ischemia is associated with decreased total blood flow, muscle microvascular perfusion as well as microvascular responses to insulin. While the recovery in muscle microvascular perfusion precedes that of total limb blood flow, recovery in muscle microvascular response to insulin is delayed. These changes may contribute to muscle injury in patients with diabetes and the muscle microvasculature is potentially a therapeutic target for PAD. Disclosure N. Wang: None. Z. Liu: None. Funding American Diabetes Association (1-17-ICTS-059 to Z.L.); National Institutes of Health (R01DK102359)

Muscle Metaboreflex Induced Increases in Effective Arterial Elastance

Dynamic exercise elicits robust changes in sympathetic activity in part due to accumulation of metabolites within the active skeletal muscle which activates group III and IV afferents triggering the muscle metaboreflex. This reflex raises cardiac output via tachycardia, increased ventricular contractility, and central blood volume mobilization which improves perfusion to the active skeletal muscle. An important component of maintaining exercise performance is optimal ventricular ‐ vascular coupling which can change with exercise, aging, as well as pathophysiological conditions. The effect of muscle metaboreflex activation (MMA) on ventricular ‐ vascular interactions is unknown. We hypothesized that MMA increases Effective Arterial Elastance (Ea). We utilized two previously published methods of evaluating Ea (End Systolic Pressure / Stroke Volume (M1) and Heart Rate x Vascular Resistance (M2)) at rest, during mild treadmill exercise, and during MMA induced via partial reductions in hindlimb blood flow imposed during exercise. At rest Ea averaged 3.0 ± 0.1 and 3.0 ± 0.2 mmHg/mL for M1 and M2, respectively. With the transition from rest to mild exercise little change in Ea occurred, whereas with subsequent MMA, Ea increased significantly by 33.6% ± 2.7 and 20.5% ± 2.3 for M1 and M2. We conclude that MMA elicits robust increases in Ea which parallel the substantial increases in ventricular performance thereby optimizing ventricular ‐ vascular coupling.Support or Funding InformationSupported by HL‐55473, HL‐126706, HL120822 and R25 GM058905This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exaggerated Pressor Response to Intermittent Contraction in Type 1 Diabetic Rats

Type 1 diabetes mellitus (T1DM) is associated with an impaired regulation of autonomic and cardiovascular responses during exercise, an effect attributable, in part, to the exercise pressor reflex. Previously, we have found that the pressor responses to both static muscle contraction and to tendon stretch are exaggerated in the early stage of T1DM. However, it is not known whether a similar response is present during dynamic exercise. Moreover, other studies have suggested that endothelial dysfunction manifests early in T1DM, which may impair muscle perfusion during exercise leading to an increased stimulation of the exercise pressor reflex. Therefore, the purpose of this study was to test whether T1DM rats have an exaggerated pressor and an attenuated blood flow response to intermittent contraction. To do this, we measured the exercise pressor reflex in unanesthetized, decerebrated T1DM (Streptozotocin 50mg/kg i.p; STZ: body weight: 328 ± 39g) and healthy (CTL: body weight: 264 ± 17g, n=5) Sprague Dawley rats by intermittently contracting the hindlimb for 30 s while continuously measuring mean arterial pressure (MAP), heart rate (HR) and popliteal arterial blood flow (BF). Popliteal vascular conductance was estimated by dividing popliteal BF by MAP. BF and VC are reported as mean change from baseline.Muscle contraction significantly increased peak MAP (STZ: 32 ± 4 mmHg, n=3; CTL: 17 ± 4 mmHg, n=5, p<0.05) but not HR (STZ: 20 ± 4 mmHg, n=3; CTL: 14 ± 6 mmHg, n=5, p>0.05) to similar developed tensions (p>0.05) in T1DM rats compared to CTL rats. Muscle contraction increased hindlimb BF (STZ: 858 ± 379 μl/min, n=3; CTL: 220 ± 40 μl/min, n=2) and hindlimb vascular conductance (STZ: 7.4 ± 4.1 μl/min/mmHg, n=3; CTL: 2.2 ± 0.3 μl/min/mmHg, n=2) from baseline in STZ and CTL rats, however, this was not significantly different between groups (p>0.05). These findings are consistent with our previous studies that found that early stage T1DM rats had an exaggerated pressor response to static muscle contraction. Furthermore, the finding that blood flow delivery was not impaired in the T1DM rats may suggest that a mismatch between metabolic demand and oxygen delivery is not contributing to the exaggerated response.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ameliorating effects of Cuscuta chinensis Lamak extract on hind‑limb ischemia, and angiogenic‑ or inflammatory associated factors in ovariectomized mice.

Cuscutachinensis Lamak (CCL) has traditionally been used in Korea to treat sexual disorders and skin problems. The aim of the present study was to investigate the effects of CCL extract on surgical injury‑induced ischemia in the hind limbs of mice. Specifically, female C57BL/6 mice were ovariectomized, and their hind‑limb vessels were ligated with surgical silk (6‑0) and excised. CCL (150or450mg/kg/BW) was then administered to the mice for 3weeks, and the blood flow rate was evaluated using a laser Doppler system at ‑7,0,7,14 and21days following hind‑limb ischemia. The serum expression profiles of angiogenic and inflammatory mediators were measured using an antibody array, and the transcript levels were reverse transcription‑quantitative polymerase chain reaction. The rate of hind limb blood flow was normalized to non‑ischemic lesions and revealed to be markedly elevated at 14 and 21days following ischemia when compared with the vehicle group. The density of capillaries in the hind limbs was also significantly increased following treatment with CCL in a dose‑dependent manner. In addition, the transcriptional expression of angiogenetic factors were upregulated, whereas that of inflammatory cytokines were downregulated. Finally, vascular endothelial cell migration and tube formation were evaluated invitro using human umbilical vein endothelial cells (HUVECs) and identified to be significantly increased following treatment with CCL. Overall, the results of the present study indicate that CCL extract exhibits therapeutic potential for the treatment of hind‑limb ischemia as it promotes peripheral angiogenic and anti‑inflammatory effects in mice.

NADPH oxidase 2 inhibitors CPP11G and CPP11H attenuate endothelial cell inflammation & vessel dysfunction and restore mouse hind-limb flow

First described as essential to the phagocytic activity of leukocytes, Nox2-derived ROS have emerged as mediators of a range of cellular and tissue responses across species from salubrious to deleterious consequences. Knowledge of their role in inflammation is limited, however. We postulated that TNFα-induced endothelial reactive oxygen species (ROS) generation and pro-inflammatory signaling would be ameliorated by targeting Nox2. Herein, we in silico-modelled two first-in-class Nox2 inhibitors developed in our laboratory, explored their cellular mechanism of action and tested their efficacy in in vitro and mouse in vivo models of inflammation. Our data show that these inhibitors (CPP11G and CPP11H) disrupted canonical Nox2 organizing factor, p47phox, translocation to Nox2 in the plasma membrane; and abolished ROS production, markedly attenuated stress-responsive MAPK signaling and downstream AP-1 and NFκB nuclear translocation in human cells. Consequently, cell adhesion molecule expression and monocyte adherence were significantly inhibited by both inhibitors. In vivo, TNFα-induced ROS and inflammation were ameliorated by targeted Nox2 inhibition, which, in turn, improved hind-limb blood flow. These studies identify a proximal role for Nox2 in propagated inflammatory signaling and support therapeutic value of Nox2 inhibitors in inflammatory disease.

Magnesium lithospermate B ameliorates microcirculation perfusion in rats by promoting vascular NO production via activating the PI3K/AKT pathway.

Microcirculation morphologically refers to the blood flow in vessels of less than 150 μm in diameter, including arterioles, capillaries and venules, which provides nutrients and removes metabolic byproducts within tissues. Microcirculation dysfunction is involved in the pathological progress of many diseases, such as obesity, hypertension, and insulin resistance. In this study we investigated the effects of magnesium lithospermate B (MLB), an active compound of the traditional Chinese medicine Slavia miltiorrhiza, on the microcirculation dysfunction in rats and the underlying molecular mechanisms. The effects of MLB on microcirculation were assessed in vivo by measuring the hindlimb blood perfusion in dextran-induced microcirculation dysfunction rats and mesentery blood flow in anesthetized rats. We demonstrated that administration of MLB restored the impaired rat hindlimb blood flow and promoted the mesenteric micoperfusion in vivo. We further revealed in these two animal models that MLB treatment significantly increased the production of total nitrite in vascular tissues (mesentery, aorta, and heart), which was confirmed in human microvascular endothelial cells (HMEC-1) treated with MLB in vitro. Moreover, we showed that MLB treatment significantly increased the phosphorylation of endothelium nitric oxide synthase (eNOS) via inducing AKT phosphorylation in vivo and in vitro. Co-administration of the eNOS inhibitor L-NAME (20 mg/kg) abolished the protective effects of MLB against dextran-induced microcirculation dysfunction in rats, whereas pretreatment with PI3K inhibitor LY294002 (10 μM) prevented eNOS activation in MLB-treated HMEC-1 cells. Our results suggest that MLB can restore the microcirculation dysfunction via activating eNOS, and in turn enhancing the vascular nitric oxide production, which is medicated by MLB-caused activation of the PI3K/AKT pathway.

Repetitive remote occlusion (RRO) stimulates eNOS-dependent blood flow and collateral expansion in hindlimb ischemia

ObjectiveCollateral expansion is an important compensatory mechanism to alleviate tissue ischemia after arterial occlusion. We investigated the efficacy and mechanisms of temporary remote hindlimb occlusion to stimulate contralateral blood flow and collateral expansion after hindlimb ischemia in mice and evaluated translation to peripheral artery disease in humans. Methods and resultsWe induced unilateral hindlimb ischemia via femoral artery excision in mice. We studied central hemodynamics, blood flow, and perfusion of the ischemic hindlimb during single and repetitive remote occlusion (RRO) of the contralateral non-ischemic hindlimb with a pressurized cuff. Similar experiments were performed in patients with unilateral peripheral artery disease (PAD). Contralateral occlusion of the non-ischemic hindlimb led to an acute increase in blood flow to the ischemic hindlimb without affecting central blood pressure and cardiac output. The increase in blood flow was sustained even after deflation of the pressure cuff. RRO over 12 days (8/day, each 5 min) led to significantly increased arterial inflow, lumen expansion of collateral arteries, and increased perfusion of the chronically ischemic hindlimb as compared to control. In NOS3-/- and after inhibition of NOS (L-NAME), and NO (ODQ), the acute and chronic effects of contralateral occlusion were abrogated and stimulation of guanylyl cyclase with cinaciguate exhibited a similar response as RRO and was not additive. Pilot studies in PAD patients demonstrated that contralateral occlusion increased arterial inflow to ischemic limbs and improved walking distance. ConclusionsRepetitive remote contralateral occlusion stimulates arterial inflow, perfusion, and functional collateral expansion in chronic hindlimb ischemia via an eNOS-dependent mechanism underscoring the potential of remote occlusion as a novel treatment option in peripheral artery disease.

Altered arterial baroreflex-muscle metaboreflex interaction in heart failure.

Two powerful reflexes controlling cardiovascular function during exercise are the muscle metaboreflex and arterial baroreflex. In heart failure (HF), the strength and mechanisms of these reflexes are altered. Muscle metaboreflex activation (MMA) in normal subjects increases mean arterial pressure (MAP) primarily via increases in cardiac output (CO), whereas in HF the mechanism shifts to peripheral vasoconstriction. Baroreceptor unloading increases MAP via peripheral vasoconstriction, and this pressor response is blunted in HF. Baroreceptor unloading during MMA in normal animals elicits an enormous pressor response via combined increases in CO and peripheral vasoconstriction. The mode of interaction between these reflexes is intimately dependent on the parameter (e.g., MAP and CO) being investigated. The interaction between the two reflexes when activated simultaneously during dynamic exercise in HF is unknown. We activated the muscle metaboreflex in chronically instrumented dogs during mild exercise (via graded reductions in hindlimb blood flow) followed by baroreceptor unloading [via bilateral carotid occlusion (BCO)] before and after induction of HF. We hypothesized that BCO during MMA in HF would cause a smaller increase in MAP and a larger vasoconstriction of ischemic hindlimb vasculature, which would attenuate the restoration of blood flow to ischemic muscle observed in normal dogs. We observed that BCO during MMA in HF increases MAP by substantial vasoconstriction of all vascular beds, including ischemic active muscle, and that all cardiovascular responses, except ventricular function, exhibit occlusive interaction. We conclude that vasoconstriction of ischemic active skeletal muscle in response to baroreceptor unloading during MMA attenuates restoration of hindlimb blood flow. NEW & NOTEWORTHY We found that baroreceptor unloading during the muscle metaboreflex in heart failure results in occlusive interaction (except for ventricular function) with significant vasoconstriction of all vascular beds. In addition, restoration of blood flow to ischemic active muscle, via preferentially larger vasoconstriction of nonischemic beds, is significantly attenuated in heart failure.

An Isolated Limb Infusion Method Allows for Broad Distribution of rAAVrh74.MCK.GALGT2 to Leg Skeletal Muscles in the Rhesus Macaque

Recombinant adeno-associated virus (rAAV)rh74.MCK.GALGT2 is a muscle-specific gene therapy that is being developed to treat forms of muscular dystrophy. Here we report on an isolated limb infusion technique in a non-human primate model, where hindlimb blood flow is transiently isolated using balloon catheters to concentrate vector in targeted leg muscles. A bilateral dose of 2.5 × 1013 vector genomes (vg)/kg/limb was sufficient to induce GALGT2-induced glycosylation in 10%–60% of skeletal myofibers in all leg muscles examined. There was a 19-fold ± 6-fold average limb-wide increase in vector genomes per microgram genomic DNA at a bilateral dose of 2.5 × 1013 vg/kg/limb compared with a bilateral dose of 6 × 1012 vg/kg/limb. A unilateral dose of 6 × 1013 vg/kg/limb showed a 12- ± 3-fold increase in treated limb muscles compared to contralateral untreated limb muscles, which received vector only after release into the systemic circulation from the treated limb. Variability in AAV biodistribution between different segments of the same muscle was 125% ± 18% for any given dose, while variability between the same muscle for any given treatment dose was 45% ± 7%. These experiments demonstrate that treatment of muscles throughout the leg with rAAVrh74.MCK.GALGT2 can be accomplished safely using an isolated limb infusion technique, where balloon catheters transiently isolate the limb vasculature, but that intra- and inter-muscle transduction variability is a significant issue.

Effects of CO2 water-bath treatment on blood flow and angiogenesis in ischemic hind limb of diabetic rat.

The effects of CO2 water-bath therapy on the hind limb of diabetic animals with or without peripheral ischemia were examined. Diabetes was induced in rats by administering streptozotocin (65 mg·kg-1), and the animals were then divided into 3 groups. After 4 weeks, peripheral ischemia was induced by ligation of the femoral artery for 2 weeks in 2 groups (diabetic ischemic) of diabetic rats, whereas the femoral artery was not occluded in the third group (diabetic). All these animals were subjected to water-bath therapy (with or without CO2 mixing; 20 min·day-1 for 5 days·week-1) for a period of 4 weeks. Both peak and mean flows, unlike minimal flow, in diabetic ischemic limbs were increased about a twofold by CO2 water-bath treatment. Morphological examination of hind limb tissue sections revealed about a twofold increase in the small artery count in diabetic ischemic animals upon CO2 water-bath treatment. These results indicate that CO2 water-bath therapy augments the blood flow and development of angiogenesis in the skeletal muscle of diabetic ischemic animals and thus may be of some benefit for the treatment of peripheral arterial disease in diabetes.

Abstract 133: Heterozygous Deletion of Transferrin Receptor 1 Suppresses Angiogenesis in a Mouse Model of Hind Limb Ischemia

Objective: Angiogenesis can be triggered under conditions of ischemia and plays a protective role in peripheral artery disease. Iron is an essential trace mineral in the living body whose intracellular metabolism is regulated by transferrin receptor 1 (TfR1). Excess iron causes tissue damage and has been reported in patients with peripheral artery disease. Therefore, we hypothesized that TfR1 contributes to the pathophysiology of hind limb ischemia through iron overload. The aim of this study is to examine whether TfR1 deletion effects angiogenesis in hind limb ischemia. Methods and Results: Since homozygous TfR1 deletion is embryonically lethal, mice with heterozygous TfR1 deletion and their wild type littermates were used in this study. To induce hind limb ischemia, the left femoral artery in 8 - 9 week-old male mice was ligated and stripped. The non-ligated right hind limb was used as a control. Blood flow was measured by laser Doppler blood flowmetry at both a baseline of 1 hour and 28 days after the surgery. In wild type, the ratio of ischemc/non-ischemic hind limb blood flow was significantly decreased to 12% at baseline, and recovered to 62% at day 28. On the other hand, TfR1 deleted mice also showed similar reduction of blood flow at baseline, whereas blood flow recovery at day 28 was attenuated to 42%. Subsequently, both right and left adductor muscles were harvested for Western blot and immunohistochemistry. TfR1 expression in the adductor muscle was decreased in TfR1 deleted mice compared to wild type, regardless of the ligation. Ferritin, a marker for iron storage, was 10 times higher in ischemic muscles than in wild type non-ischemic muscles. In contrast, ferritin levels in TfR1 deleted mice were not altered by the surgery. Finally, immunohistochemistry for CD31 was performed to evaluate angiogenesis. The ischemic adductor muscle of TfR1 deleted mice had few CD31 positive vascular structures. Conclusions: Heterozygous deletion of TfR1 attenuated iron overload and angiogenesis in a mouse model of hind limb ischemia. TfR1 may be a novel therapeutic target for the treatment of peripheral artery disease.

Muscle Metaboreflex Control of Left Ventricular Filling Pressure

When oxygen delivery to active skeletal muscle is limited, afferent signals from the muscle elicit reflex increases in cardiac output (CO) and mean arterial pressure (MAP) in order to increase perfusion to the ischemic muscle ‐ termed the muscle metaboreflex (MMR). Previous studies have shown that in normal subjects, CO is increased predominantly through increases in heart rate (HR) while maintaining stroke volume (SV) essentially constant. However, increases in HR alone have limited ability to raise CO because as HR increases, SV falls due to decreased filling time. Increases in ventricular contractility can limit the falls in SV, however with sustained increases in CO, blood is translocated from the venous to the arterial circulation and ventricular filling pressure falls which limits the ability to maintain elevated CO. To sustain increases in CO, central blood volume mobilization must also occur to maintain ventricular filling pressure. We previously showed that MMR activation does increase central venous pressure (Sheriff, Augustyniak and O'Leary, Am. J. Physiol., 1998), but whether this translates into increases in left ventricular end‐diastolic pressure (LVEDP) is unknown. MMR was activated via reductions in hindlimb blood flow during mild treadmill exercise in chronically instrumented canines before and after induction of heart failure (HF, rapid ventricular pacing, 220–240 bpm, ~30 days). In normal animals, MMR activation induced a rise in LVEDP from 14.4 ± 1.8 to 18.2 ± 1.8 mmHg (p<0.05) despite substantial increases in HR (Δ 26 ± 4 bpm), CO (Δ 1.45 ± 0.16 l/min) and MAP (Δ 42.3 ± 2.6 mmHg). After induction of HF, MMA activation increased LVEDP from 28.6 ± 2.5 to 38.7 ± 2.6 mmHg (p<0.05) which was a significantly larger rise than observed in normal animals. In HF, the attenuated rise in CO (Δ 0.20 ± 0.08 l/min) with MMR activation likely contributes to the exaggerated increase in LVEDP. We conclude that MMR activation elicits significant blood volume mobilization which increases cardiac filling pressure which thereby likely helps sustain SV despite shortened filling time. In HF, the limited ability to raise CO is not due to reduced filling pressures.Support or Funding InformationNIH RO1 Grants HL055473 and HL126706This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuronal nitric oxide synthase regulation of skeletal muscle functional hyperemia: exercise training and moderate compensated heart failure

Nitric oxide (NO) modulates oxygen delivery-utilization matching in resting and contracting skeletal muscle. Recent reports indicate that neuronal NO synthase (nNOS)-mediated vasoregulation during contractions is enhanced with exercise training and impaired with chronic heart failure (HF). Consequently, we tested the hypothesis that selective nNOS inhibition (S-methyl-l-thiocitrulline; SMTC, 2.1 μmol/kg) would produce attenuated reductions in muscle blood flow during moderate/heavy submaximal exercise in sedentary HF rats compared to their healthy counterparts. In addition, SMTC was expected to evoke greater reductions in exercising muscle blood flow in trained compared to sedentary healthy and HF rats. Blood flow during submaximal treadmill running (20 min/m, 5% grade) was determined via radiolabeled microspheres pre- and post-SMTC administration in healthy sedentary (Healthy + Sed, n = 8), healthy exercise trained (Healthy + ExT, n = 8), HF sedentary (HF + Sed, left ventricular end-diastolic pressure (LVEDP) = 12 ± 1 mmHg, n = 8), and HF exercise trained (HF + ExT, LVEDP = 16 ± 2 mmHg, n = 7) rats. nNOS contribution to exercising total hindlimb blood flow (ml/min/100 g) was not increased by training in either healthy or HF groups (Healthy + Sed: 105 ± 11 vs. 108 ± 16; Healthy + ExT: 96 ± 9 vs. 91 ± 7; HF + Sed: 124 ± 6 vs. 110 ± 12; HF + ExT: 107 ± 13 vs. 101 ± 8; control vs. SMTC, respectively; p > .05 for all). Similarly, SMTC did not reduce exercising blood flow in the majority of individual hindlimb muscles in any group (p > .05 for all, except for the semitendinosus and adductor longus in HF + Sed and the adductor longus in HF + ExT; p < .05). Contrary to our hypothesis, we find no support for either upregulation of nNOS function contributing to exercise hyperemia after training or its dysregulation with chronic HF.

Carthamus tinctorius L. extract improves hemodynamic and vascular alterations in a rat model of renovascular hypertension through Ang II-AT1R-NADPH oxidase pathway

Carthamus tinctorius L. (CT) is widely used in Asian countries as a beverage and in folk medicine. The effects of CT extract on hemodynamics, vascular remodeling, the renin-angiotensin system (RAS) and oxidative stress in the two-kidney, one clip (2K-1C) hypertensive rat model were investigated. Renovascular hypertension was induced in male Sprague-Dawley rats and were treated with CT extract (500mg/kg/day) or captopril (5mg/kg/day) or vehicle for four weeks. CT extract or captopril reduced blood pressure, hindlimb vascular resistance, and increased hindlimb blood flow in 2K-1C hypertensive rats (p<0.05). Increases in aortic wall thickness, cross-sectional area and collagen deposition in 2K-1C rats were alleviated with CT extract or captopril treatment (p<0.05). CT extract or captopril suppressed RAS activation, including elevated serum ACE activity, and plasma Ang II level and up-regulated aortic AT1R protein expression in 2K-1C rats (p<0.05). Furthermore, CT extract or captopril reduced vascular superoxide production, aortic NADPH oxidase subunit gp91phox expression and increased plasma nitric oxide metabolite levels in 2K-1C rats (p<0.05). These findings suggest that CT extract ameliorated hemodynamic alteration and vascular remodeling in 2K-1C hypertensive rats. Possible mechanisms may involve RAS inhibitor effects and potent antioxidant activity.

Exenatide Improves Bone Quality in a Murine Model of Genetically Inherited Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is associated with skeletal complications, including an increased risk of fractures. Reduced blood supply and bone strength may contribute to this skeletal fragility. We hypothesized that long-term administration of Exenatide, a glucagon-like peptide-1 receptor agonist, would improve bone architecture and strength of T2DM mice by increasing blood flow to bone, thereby stimulating bone formation. In this study, we used a model of obesity and severe T2DM, the leptin receptor-deficient db/db mouse to assess alterations in bone quality and hindlimb blood flow and to examine the beneficial effects of 4 weeks administration of Exenatide. As expected, diabetic mice showed marked alterations in bone structure, remodeling and strength, and basal vascular tone compared with lean mice. Exenatide treatment improved trabecular bone mass and architecture by increasing bone formation rate, but only in diabetic mice. Although there was no effect on hindlimb perfusion at the end of this treatment, Exenatide administration acutely increased tibial blood flow. While Exenatide treatment did not restore the impaired bone strength, intrinsic properties of the matrix, such as collagen maturity, were improved. The effects of Exenatide on in vitro bone formation were further investigated in primary osteoblasts cultured under high-glucose conditions, showing that Exenatide reversed the impairment in bone formation induced by glucose. In conclusion, Exenatide improves trabecular bone mass by increasing bone formation and could protect against the development of skeletal complications associated with T2DM.

Skeletal muscle protein accretion rates and hindlimb growth are reduced in late gestation intrauterine growth-restricted fetal sheep.

Adults who were affected by intrauterine growth restriction (IUGR) suffer from reductions in muscle mass, which may contribute to insulin resistance and the development of diabetes. We demonstrate slower hindlimb linear growth and muscle protein synthesis rates that match the reduced hindlimb blood flow and oxygen consumption rates in IUGR fetal sheep. These adaptations resulted in hindlimb blood flow rates in IUGR that were similar to control fetuses on a weight-specific basis. Net hindlimb glucose uptake and lactate output rates were similar between groups, whereas amino acid uptake was significantly lower in IUGR fetal sheep. Among all fetuses, blood O2 saturation and plasma glucose, insulin and insulin-like growth factor-1 were positively associated and norepinephrine was negatively associated with hindlimb weight. These results further our understanding of the metabolic and hormonal adaptations to reduced oxygen and nutrient supply with placental insufficiency that develop to slow hindlimb growth and muscle protein accretion. Reduced skeletal muscle mass in the fetus with intrauterine growth restriction (IUGR) persists into adulthood and may contribute to increased metabolic disease risk. To determine how placental insufficiency with reduced oxygen and nutrient supply to the fetus affects hindlimb blood flow, substrate uptake and protein accretion rates in skeletal muscle, late gestation control (CON) (n=8) and IUGR (n=13) fetal sheep were catheterized with aortic and femoral catheters and a flow transducer around the external iliac artery. Muscle protein kinetic rates were measured using isotopic tracers. Hindlimb weight, linear growth rate, muscle protein accretion rate and fractional synthetic rate were lower in IUGR compared to CON (P<0.05). Absolute hindlimb blood flow was reduced in IUGR (IUGR: 32.9±5.6mlmin-1 ; CON: 60.9±6.5mlmin-1 ; P<0.005), although flow normalized to hindlimb weight was similar between groups. Hindlimb oxygen consumption rate was lower in IUGR (IUGR: 10.4±1.4μmolmin-1 100g-1 ; CON: 14.7±1.3μmolmin-1 100g-1 ; P<0.05). Hindlimb glucose uptake and lactate output rates were similar between groups, whereas amino acid uptake was lower in IUGR (IUGR: 1.3±0.5μmolmin-1 100g-1 ; CON: 2.9±0.2μmolmin-1 100g-1 ; P<0.05). Blood O2 saturation (r2 =0.80, P<0.0001) and plasma glucose (r2 =0.68, P<0.0001), insulin (r2 =0.40, P<0.005) and insulin-like growth factor (IGF)-1 (r2 =0.80, P<0.0001) were positively associated and norepinephrine (r2 =0.59, P<0.0001) was negatively associated with hindlimb weight. Slower hindlimb linear growth and muscle protein synthesis rates match reduced hindlimb blood flow and oxygen consumption rates in the IUGR fetus. Metabolic adaptations to slow hindlimb growth are probably hormonally-mediated by mechanisms that include increased fetal norepinephrine and reduced IGF-1 and insulin.

Muscle metaboreflex-induced vasoconstriction in the ischemic active muscle is exaggerated in heart failure.

When oxygen delivery to active muscle is insufficient to meet the metabolic demand during exercise, metabolites accumulate and stimulate skeletal muscle afferents, inducing a reflex increase in blood pressure, termed the muscle metaboreflex. In healthy individuals, muscle metaboreflex activation (MMA) during submaximal exercise increases arterial pressure primarily via an increase in cardiac output (CO), as little peripheral vasoconstriction occurs. This increase in CO partially restores blood flow to ischemic muscle. However, we recently demonstrated that MMA induces sympathetic vasoconstriction in ischemic active muscle, limiting the ability of the metaboreflex to restore blood flow. In heart failure (HF), increases in CO are limited, and metaboreflex-induced pressor responses occur predominantly via peripheral vasoconstriction. In the present study, we tested the hypothesis that vasoconstriction of ischemic active muscle is exaggerated in HF. Changes in hindlimb vascular resistance [femoral arterial pressure ÷ hindlimb blood flow (HLBF)] were observed during MMA (via graded reductions in HLBF) during mild exercise with and without α1-adrenergic blockade (prazosin, 50 µg/kg) before and after induction of HF. In normal animals, initial HLBF reductions caused metabolic vasodilation, while reductions below the metaboreflex threshold elicited reflex vasoconstriction, in ischemic active skeletal muscle, which was abolished after α1-adrenergic blockade. Metaboreflex-induced vasoconstriction of ischemic active muscle was exaggerated after induction of HF. This heightened vasoconstriction impairs the ability of the metaboreflex to restore blood flow to ischemic muscle in HF and may contribute to the exercise intolerance observed in these patients. We conclude that sympathetically mediated vasoconstriction of ischemic active muscle during MMA is exaggerated in HF. NEW & NOTEWORTHY We found that muscle metaboreflex-induced vasoconstriction of the ischemic active skeletal muscle from which the reflex originates is exaggerated in heart failure. This results in heightened metaboreflex activation, which further amplifies the reflex-induced vasoconstriction of the ischemic active skeletal muscle and contributes to exercise intolerance in patients.

Natural compound bavachalcone promotes the differentiation of endothelial progenitor cells and neovascularization through the RORα-erythropoietin-AMPK axis.

In cardiovascular diseases, endothelial function is impaired and the level of circulating endothelial progenitor cells (EPCs) is low. This study investigated whether the natural bioactive component bavachalcone (BavaC) induces the differentiation of EPCs and neovascularization in vivo; the underlying mechanisms were also examined. We observed that the treatment of rat bone marrow–derived cells with a very low dose of BavaC significantly promoted EPC differentiation. In our hindlimb ischemia models, low–dose BavaC administered orally for 14 days stimulated the recovery of ischemic hindlimb blood flow, increased circulating EPCs, and promoted capillary angiogenesis. The BavaC treatment of rat bone marrow cells for 24 h initiated the AMP–activated protein kinase (AMPK) activity required for the differentiation of EPCs. Further testing revealed that BavaC and CGP52608, a retinoic acid receptor–related orphan receptor α (RORα) activator, enhanced the activity of RORα1 and EPO luciferase reporter gene. BavaC treatment also elevated EPO mRNA and protein expression in vitro and in vivo and the circulating EPO levels in rats. By contrast, the RORα antagonist VPR66 inhibited BavaC–induced EPO reporter activity, and differentiation of bone marrow cells into endothelial progenitor cells. Overall, this study revealed that BavaC promotes EPC differentiation and neovascularization through a RORα–EPO–AMPK axis. BavaC can be used as a promising angiogenesis agent for enhancing angiogenesis and tissue repair.

Mitochondrial-Targeted Antioxidant Maintains Blood Flow, Mitochondrial Function, and Redox Balance in Old Mice Following Prolonged Limb Ischemia.

Aging is a major factor in the decline of limb blood flow with ischemia. However, the underlying mechanism remains unclear. We investigated the role of mitochondrial reactive oxygen species (ROS) with regard to limb perfusion recovery in aging during ischemia. We performed femoral artery ligation in young and old mice with or without treatment with a scavenger of mitochondrial superoxide, MitoTEMPO (180 μg/kg/day, from pre-operative day 7 to post-operative day (POD) 21) infusion using an implanted mini-pump. The recoveries of cutaneous blood flow in the ischemic hind limb were lower in old mice than in young mice but were improved in MitoTEMPO-treated old mice. Mitochondrial DNA damage appeared in ischemic aged muscles but was eliminated by MitoTEMPO treatment. For POD 2, MitoTEMPO treatment suppressed the expression of p53 and the ratio of Bax/Bcl2 and upregulated the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in ischemic aged skeletal muscles. For POD 21, MitoTEMPO treatment preserved the expression of PGC-1α in ischemic aged skeletal muscle. The ischemic soleus of old mice showed a lower mitochondrial respiratory control ratio in POD 21 compared to young mice, which was recovered in MitoTEMPO-treated old mice. Scavenging of mitochondrial superoxide attenuated mitochondrial DNA damage and preserved the mitochondrial respiration, in addition to suppression of the expression of p53 and preservation of the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) in ischemic skeletal muscles with aging. Resolution of excessive mitochondrial superoxide could be an effective therapy to recover blood flow of skeletal muscle during ischemia in senescence.

Intracellular adenosine regulates epigenetic programming in endothelial cells to promote angiogenesis

The nucleoside adenosine is a potent regulator of vascular homeostasis, but it remains unclear how expression or function of the adenosine‐metabolizing enzyme adenosine kinase (ADK) and the intracellular adenosine levels influence angiogenesis. We show here that hypoxia lowered the expression of ADK and increased the levels of intracellular adenosine in human endothelial cells. Knockdown (KD) of ADK elevated intracellular adenosine, promoted proliferation, migration, and angiogenic sprouting in human endothelial cells. Additionally, mice deficient in endothelial ADK displayed increased angiogenesis as evidenced by the rapid development of the retinal and hindbrain vasculature, increased healing of skin wounds, and prompt recovery of arterial blood flow in the ischemic hindlimb. Mechanistically, hypomethylation of the promoters of a series of pro‐angiogenic genes, especially for VEGFR2 in ADK KD cells, was demonstrated by the Infinium methylation assay. Methylation‐specific PCR, bisulfite sequencing, and methylated DNA immunoprecipitation further confirmed hypomethylation in the promoter region of VEGFR2 in ADK‐deficient endothelial cells. Accordingly, loss or inactivation of ADK increased VEGFR2 expression and signaling in endothelial cells. Based on these findings, we propose that ADK downregulation‐induced elevation of intracellular adenosine levels in endothelial cells in the setting of hypoxia is one of the crucial intrinsic mechanisms that promote angiogenesis.