ABSTRACT
BACKGROUND: Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signaling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation. METHODS: Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for 10 minutes after intravenous injection of 2×108 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or KATP channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease. RESULTS: Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of endothelial nitric oxide synthase abolished the effects of therapeutic ultrasound, indicating downstream signaling through both nitric oxide and prostaglandins. CONCLUSIONS: Therapeutic ultrasound using microbubble cavitation to increase muscle perfusion relies on shear-dependent increases in ATP, which can act through a diverse portfolio of purinergic signaling pathways. These events can reverse hindlimb ischemia in mice for >24 hours and increase muscle blood flow in patients with sickle cell disease. CLINICAL TRIAL REGISTRATION: URL: http://clinicaltrials.gov. Unique identifier: NCT01566890.
Subject(s)
Adenosine Triphosphate/metabolism , Muscle, Skeletal/blood supply , Purinergic Agents/metabolism , Ultrasonography/methods , Animals , Hemodynamics , Humans , Male , Mice , Mice, Inbred C57BL , Microbubbles , Signal TransductionABSTRACT
PURPOSE: Our aim was to determine whether pharmacologic vasodilation is an alternative to exercise stress during limb perfusion imaging for peripheral artery disease (PAD). METHODS: Quantitative contrast-enhanced ultrasound (CEU) perfusion imaging of the bilateral anterior thigh and calf was performed in nine control subjects and nine patients with moderate to severe PAD at rest and during vasodilator stress with dipyridamole. For those who were able, CEU of the calf was then performed during modest plantar flexion exercise (20 watts). CEU time-intensity data were analyzed to quantify microvascular blood flow (MBF) and its parametric components of microvascular blood volume and flux rate. RESULTS: Thigh and calf skeletal muscle MBF at rest was similar between control and PAD patients. During dipyridamole, MBF increased minimally (Subject(s)
Dipyridamole/pharmacology
, Extremities/blood supply
, Muscle, Skeletal/blood supply
, Perfusion Imaging/methods
, Peripheral Arterial Disease/diagnosis
, Regional Blood Flow/physiology
, Ultrasonography/methods
, Aged
, Ankle Brachial Index
, Blood Flow Velocity/physiology
, Contrast Media/pharmacology
, Exercise Test/methods
, Female
, Humans
, Male
, Middle Aged
, Peripheral Arterial Disease/physiopathology
, Vasodilator Agents/pharmacology
ABSTRACT
KEY POINTS: In fetuses, chronic anaemia stimulates cardiac growth; simultaneously, blood flow to the heart muscle itself is increased, and reserve blood flow capacity of the coronary vascular bed is preserved. Here we examined functional adaptations of the capillaries and small blood vessels responsible for delivering oxygen to the anaemic fetal heart muscle using contrast-enhanced echocardiography. We demonstrate that coronary microvascular flux rate doubled in anaemic fetuses compared to control fetuses, both at rest and during maximal flow, suggesting reduced microvascular resistance consistent with capillary widening. Cardiac fractional microvascular blood volume was not greater in anaemic fetuses, suggesting that growth of new microvascular vessels does not contribute to the increased flow per volume of myocardium. These unusual changes in microvascular function during anaemia may indicate novel adaptive strategies in the fetal heart. ABSTRACT: Fetal anaemia causes cardiac adaptations that have immediate and life-long repercussions on heart function and health. It is known that resting and maximal coronary conductance both increase during chronic fetal anaemia, but the coronary microvascular changes responsible for the adaptive response are unknown. Until recently, technical limitations have prevented quantifying functional capillary-level adaptations in the in vivo fetal heart. Our objective was to characterise functional microvascular adaptations in chronically anaemic fetal sheep. Chronically instrumented fetuses were randomized to a control group (n = 11) or were made anaemic by isovolumetric haemorrhage (n = 12) for 1 week prior to myocardial contrast echocardiography at 85% of gestation. Anaemia augmented cardiac mass by 23% without changing body weight. In anaemic fetuses, microvascular blood flow per volume of myocardium was twice that of control fetuses at rest, during vasodilatory hyperaemia, and during hyperaemia plus increased aortic pressure. The elevated blood flow was attributable almost entirely to an increase in microvascular blood flux rate whereas microvascular blood volumes were not different between groups at baseline, during hyperaemia, or with hyperaemia plus increased aortic pressure. Increased coronary microvascular flux rate in response to chronic fetal anaemia is consistent with expected reductions in capillary resistance from capillary diameter widening detected in earlier histological studies.
Subject(s)
Adaptation, Physiological , Anemia/physiopathology , Coronary Vessels/physiology , Fetal Heart/physiology , Hyperemia/etiology , Microcirculation , Pregnancy Complications/physiopathology , Anemia/complications , Animals , Blood Pressure , Capillaries/physiology , Capillaries/physiopathology , Coronary Vessels/physiopathology , Female , Fetal Heart/physiopathology , Hyperemia/physiopathology , Pregnancy , SheepABSTRACT
OBJECTIVE: Focal junctional tourniquets (JTs) have been developed to control hemorrhage from proximal limb injuries. These devices may permit greater collateral perfusion than circumferential tourniquets. We hypothesized that JTs eliminate large-vessel pulse pressure yet allow a small amount of residual limb perfusion that could be useful for maintaining tissue viability. METHODS: Ten healthy control subjects were studied. Transthoracic echocardiography, Doppler ultrasound of the femoral artery (FA) and posterior tibial artery, and contrast-enhanced ultrasound (CEU) perfusion imaging of the anterior thigh extensor and calf plantar flexor muscles were performed at baseline and during application of a JT over the common FA. Intramuscular arterial pulsatility index was also measured from CEU intensity variation during the cardiac cycle. RESULTS: FA flow was eliminated by JTs in all subjects; posterior tibial flow was eliminated in all but one. Perfusion measured in the thigh and calf muscles was similar at baseline (0.33 ± 0.29 vs 0.29 ± 0.22 mL/min/g). Application of the JT resulted in a reduction of perfusion (P < .05) that was similar for the thigh and calf (0.08 ± 0.07 and 0.10 ± 0.03 mL/min/g). On CEU, microvascular flux rate was reduced by ≈55%, and functional microvascular blood volume was reduced by ≈35%. Arterial pulsatility index was reduced by ≈90% in the calf. JT inflation did not alter left ventricle dimensions, fractional shortening, cardiac output, or arterial elastance as a measure of total systolic load. CONCLUSIONS: Application of a JT eliminates conduit arterial pulse and markedly reduces intramuscular pulse pressure, but thigh and calf skeletal muscle perfusion is maintained at 25% to 35% of basal levels. These data suggest that JTs that are used to control limb hemorrhage allow residual tissue perfusion even when pulse pressure is absent.
Subject(s)
Contrast Media , Femoral Artery/diagnostic imaging , Fluorocarbons , Hemodynamics , Hemostatic Techniques/instrumentation , Muscle, Skeletal/blood supply , Perfusion Imaging/methods , Tibial Arteries/diagnostic imaging , Tourniquets , Ultrasonography, Doppler, Color , Ultrasonography, Doppler, Pulsed , Adult , Blood Flow Velocity , Blood Pressure , Equipment Design , Female , Femoral Artery/physiology , Healthy Volunteers , Humans , Lower Extremity , Male , Microcirculation , Middle Aged , Predictive Value of Tests , Regional Blood Flow , Tibial Arteries/physiology , Time Factors , Tissue Survival , Young AdultABSTRACT
BACKGROUND: Stem cells are thought to enhance vascular remodeling in ischemic tissue in part through paracrine effects. Using molecular imaging, we tested the hypothesis that treatment of limb ischemia with multipotential adult progenitor cells (MAPCs) promotes recovery of blood flow through the recruitment of proangiogenic monocytes. METHODS AND RESULTS: Hind-limb ischemia was produced in mice by iliac artery ligation, and MAPCs were administered intramuscularly on day 1. Optical imaging of luciferase-transfected MAPCs indicated that cells survived for 1 week. Contrast-enhanced ultrasound on days 3, 7, and 21 showed a more complete recovery of blood flow and greater expansion of microvascular blood volume in MAPC-treated mice than in controls. Fluorescent microangiography demonstrated more complete distribution of flow to microvascular units in MAPC-treated mice. On ultrasound molecular imaging, expression of endothelial P-selectin and intravascular recruitment of CX(3)CR-1-positive monocytes were significantly higher in MAPC-treated mice than in the control groups at days 3 and 7 after arterial ligation. Muscle immunohistology showed a >10-fold-greater infiltration of monocytes in MAPC-treated than control-treated ischemic limbs at all time points. Intravital microscopy of ischemic or tumor necrosis factor-α-treated cremaster muscle demonstrated that MAPCs migrate to perimicrovascular locations and potentiate selectin-dependent leukocyte rolling. In vitro migration of human CD14(+) monocytes was 10-fold greater in response to MAPC-conditioned than basal media. CONCLUSIONS: In limb ischemia, MAPCs stimulate the recruitment of proangiogenic monocytes through endothelial activation and enhanced chemotaxis. These responses are sustained beyond the MAPC lifespan, suggesting that paracrine effects promote flow recovery by rebalancing the immune response toward a more regenerative phenotype.
Subject(s)
Extremities/blood supply , Ischemia/therapy , Molecular Imaging , Neovascularization, Physiologic/physiology , Paracrine Communication/physiology , Stem Cell Transplantation , Adult Stem Cells/diagnostic imaging , Adult Stem Cells/drug effects , Adult Stem Cells/transplantation , Animals , CX3C Chemokine Receptor 1 , Cell Movement/physiology , Extremities/diagnostic imaging , Extremities/pathology , Humans , Iliac Artery/diagnostic imaging , Iliac Artery/drug effects , Iliac Artery/physiopathology , Ischemia/diagnostic imaging , Ischemia/pathology , Lipopolysaccharide Receptors/analysis , Mice , Mice, Inbred C57BL , Microvessels/diagnostic imaging , Microvessels/drug effects , Microvessels/pathology , Microvessels/physiopathology , Monocytes/pathology , Monocytes/physiology , Multipotent Stem Cells/diagnostic imaging , Multipotent Stem Cells/drug effects , Multipotent Stem Cells/transplantation , Muscle, Skeletal/blood supply , Muscle, Skeletal/diagnostic imaging , Muscle, Skeletal/pathology , Neovascularization, Physiologic/drug effects , P-Selectin/biosynthesis , Paracrine Communication/drug effects , Receptors, Chemokine/analysis , Transplantation, Heterologous , Tumor Necrosis Factor-alpha/pharmacology , UltrasonographyABSTRACT
Skeletal muscle microvascular blood flow (MBF) increases in response to physiological hyperinsulinemia. This vascular action of insulin may facilitate glucose uptake. We hypothesized that epoxyeicosatrienoic acids (EETs), a family of arachadonic, acid-derived, endothelium-derived hyperpolarizing factors, are mediators of insulin's microvascular effects. Contrast-enhanced ultrasound (CEU) was performed to quantify skeletal muscle capillary blood volume (CBV) and MBF in wild-type and obese insulin-resistant (db/db) mice after administration of vehicle or trans-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid (t-AUCB), an inhibitor of soluble epoxide hydrolase that converts EETs to less active dihydroxyeicosatrienoic acids. Similar studies were performed in rats pretreated with l-NAME. CEU was also performed in rats undergoing a euglycemic hyperinsulinemic clamp, half of which were pretreated with the epoxygenase inhibitor MS-PPOH to inhibit EET synthesis. In both wild-type and db/db mice, intravenous t-AUCB produced an increase in CBV (65-100% increase at 30 min, P < 0.05) and in MBF. In db/db mice, t-AUCB also reduced plasma glucose by â¼15%. In rats pretreated with l-NAME, t-AUCB after produced a significant ≈20% increase in CBV, indicating a component of vascular response independent of nitric oxide (NO) production. Hyperinsulinemic clamp produced a time-dependent increase in MBF (19 ± 36 and 76 ± 49% at 90 min, P = 0.026) that was mediated in part by an increase in CBV. Insulin-mediated changes in both CBV and MBF during the clamp were blocked entirely by MS-PPOH. We conclude that EETs are a mediator of insulin-mediated augmentation in skeletal muscle perfusion and are involved in regulating changes in CBV during hyperinsulinemia.
Subject(s)
8,11,14-Eicosatrienoic Acid/metabolism , Insulin/pharmacology , Muscle, Skeletal/blood supply , Muscle, Skeletal/metabolism , 8,11,14-Eicosatrienoic Acid/antagonists & inhibitors , Animals , Benzoates/pharmacology , Blood Volume/drug effects , Epoxide Hydrolases/antagonists & inhibitors , Hyperinsulinism/physiopathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microcirculation/drug effects , Muscle, Skeletal/drug effects , Rats , Rats, Sprague-Dawley , Regional Blood Flow/drug effects , Urea/analogs & derivatives , Urea/pharmacologyABSTRACT
Cardiac allograft vasculopathy (CAV) remains a common long-term complication of cardiac transplantation. While invasive coronary angiography is considered the gold standard, it is also invasive and lacks sensitivity to detect early, distal CAV. Although vasodilator stress myocardial contrast echocardiography perfusion imaging (MCE) is used in the detection of microvascular disease in non-transplant patients, there is little data guiding its use in transplant recipients. Herein is a case series of four heart transplant recipients that had vasodilator stress MCE performed in addition to invasive coronary angiography for CAV surveillance. MCE at rest and after regadenason was performed using a continuous infusion of lipid-shelled microbubbles. We describe a case of normal microvascular function, diffuse microvascular dysfunction, patchy sub-endocardial perfusion defects and a focal sub-endocardial perfusion defect. Cardiac allograft vasculopathy can be heralded by several different perfusion patterns on MCE in patients after orthotopic heart transplant. The varying prognoses and potential interventions for these different patterns require further investigation.
ABSTRACT
BACKGROUND: In heart failure with reduced ejection fraction (HFrEF), abnormal regulation of skeletal muscle perfusion contributes to reduced exercise tolerance. The aim of this study was to test the hypothesis that improvement in functional status after permanent left ventricular assist device (LVAD) implantation in patients with HFrEF is related to improvement in muscle perfusion during work, which was measured using contrast-enhanced ultrasound (CEUS). METHODS: CEUS perfusion imaging of calf muscle at rest and during low-intensity plantar flexion exercise (20 W, 0.2 Hz) was performed in patients with HFrEF (n = 22) at baseline and 3 months after placement of permanent LVADs. Parametric analysis of CEUS data was used to quantify muscle microvascular blood flow (MBF), blood volume index, and red blood cell flux rate. For subjects alive at 3 months, comparisons were made between those with New York Heart Association functional class I or II (n = 13) versus III or IV (n = 7) status after LVAD. Subjects were followed for a median of 5.7 years for mortality. RESULTS: Echocardiographic data before and after LVAD placement and LVAD parameters were similar in subjects classified with New York Heart Association functional class I-II versus functional class III-IV after LVAD. Skeletal muscle MBF at rest and during exercise before LVAD implantation was also similar between groups. After LVAD placement, resting MBF remained similar between groups, but during exercise those with New York Heart Association functional class I or II had greater exercise MBF (111 ± 60 vs 52 ± 38 intensity units/sec, P = .03), MBF reserve (median, 4.45 [3.95 to 6.80] vs 2.22 [0.98 to 3.80]; P = .02), and percentage change in exercise MBF (median, 73% [-28% to 83%] vs -45% [-80% to 26%]; P = .03). During exercise, increases in MBF were attributable to faster microvascular flux rate, with little change in blood volume index, indicating impaired exercise-mediated microvascular recruitment. The only clinical or echocardiographic feature that correlated with post-LVAD exercise MBF was a history of diabetes mellitus. There was a trend toward better survival in patients who demonstrated improvement in muscle exercise MBF after LVAD placement (P = .05). CONCLUSIONS: CEUS perfusion imaging can quantify peripheral vascular responses to advanced therapies for HFrEF. After LVAD implantation, improvement in functional class is seen in patients with improvements in skeletal muscle exercise perfusion and flux rate, implicating a change in vasoactive substances that control resistance arteriolar tone.
Subject(s)
Heart Failure , Heart-Assist Devices , Heart Failure/diagnostic imaging , Heart Failure/therapy , Humans , Muscle, Skeletal/diagnostic imaging , Perfusion , Stroke VolumeABSTRACT
BACKGROUND: Noninvasive molecular imaging of recent ischemia can potentially be used to diagnose acute coronary syndrome (ACS) with high accuracy. OBJECTIVES: The authors hypothesized that bedside myocardial contrast echocardiography (MCE) ischemic memory imaging could be achieved with phosphatidylserine microbubbles (MBPS) that are retained in the microcirculation via ischemia-associated endothelial activation. METHODS: A dose-finding study was performed in healthy volunteers (n = 17) to establish optimal MBPS dosing. Stable patients with ACS (n = 30) and confirmed antecedent but resolved myocardial ischemia were studied within 2 hours of coronary angiography and percutaneous coronary intervention (PCI) when indicated. MCE molecular imaging was performed 8 minutes after intravenous administration of MBPS. MCE perfusion imaging was used to assess the status of the postischemic microcirculation. RESULTS: Based on dose-finding studies, 0.10 or 0.15 mL of MBPS based on body mass was selected. In patients with ACS, all but 2 underwent primary PCI. MCE molecular imaging signal intensity was greater in the postischemic risk area vs remote territory (median [95% CI]: 56 [33-66] vs 8 [2-17] IU; P < 0.001) with a receiver-operating characteristic curve C-statistic of 0.94 to differentiate post-ischemic from remote territory. Molecular imaging signal in the risk area was not related to type of ACS (unstable angina: 3; non-ST-segment elevation myocardial infarction: 14; ST-segment elevation myocardial infarction: 13), peak troponin, time to PCI, post-PCI myocardial perfusion, GRACE (Global Registry of Acute Coronary Events) score, or HEART score. CONCLUSIONS: Molecular imaging with point-of-care echocardiography and MBPS can detect recent but resolved myocardial ischemia. This bedside technique requires only minutes to perform and appears independent of the degree of ischemia. (Ischemic Memory Imaging With Myocardial Contrast Echocardiography; NCT03009266).
Subject(s)
Acute Coronary Syndrome/diagnostic imaging , Acute Coronary Syndrome/physiopathology , Echocardiography/methods , Microcirculation , Molecular Imaging/methods , Myocardial Ischemia/diagnostic imaging , Myocardial Ischemia/physiopathology , Point-of-Care Systems , Adult , Aged , Algorithms , Contrast Media/pharmacology , Coronary Angiography/methods , Dose-Response Relationship, Drug , Female , Healthy Volunteers , Humans , Infusions, Intravenous , Kinetics , Male , Microbubbles , Middle Aged , Myocardial Infarction , Myocardial Perfusion Imaging , Myocardium/pathology , Percutaneous Coronary Intervention , Young AdultABSTRACT
Coronary microvascular dysfunction (MVD) is a syndrome of abnormal regulation of vascular tone, particularly during increased metabolic demand. While there are several risk factors for MVD, some of which are similar to those for coronary artery disease (CAD), the cause of MVD is not understood. We hypothesized that MVD in symptomatic non-elderly subjects would be characterized by specific lipidomic profiles. Subjects (n = 20) aged 35-60 years and referred for computed tomography coronary angiography (CTA) for chest pain but who lacked obstructive CAD (>50% stenosis), underwent quantitative regadenoson stress-rest myocardial contrast echocardiography (MCE) perfusion imaging for MVD assessment. The presence of MVD defined by kinetic analysis of MCE data was correlated with lipidomic profiles in plasma measured by liquid chromatography and high-resolution mass spectrometry. Nine of twenty subjects had evidence of MVD, defined by reduced hyperemic perfusion versus other subjects (beta-value 1.62 ± 0.44 vs. 2.63 ± 0.99 s-1, p = 0.009). Neither the presence of high-risk but non-obstructive CAD on CTA, nor CAD risk factors were different for those with versus without MVD. Lipidomic analysis revealed that patients with MVD had lower concentrations of long-carbon chain triacylglycerols and diacylglycerols, and higher concentrations of short-chain triacylglycerols. The diacylglycerol containing stearic and linoleic acid classified all participants correctly. We conclude that specific lipidomic plasma profiles occur in MVD involving saturated long-chain fatty acid-containing acylglycerols that are distinctly different from those in non-obstructive CAD. These patterns could be used to better characterize the pathobiology and potential treatments for this condition.
ABSTRACT
OBJECTIVES: The authors investigated ideal acoustic conditions on a clinical scanner custom-programmed for ultrasound (US) cavitation-mediated flow augmentation in preclinical models. We then applied these conditions in a first-in-human study to test the hypothesis that contrast US can increase limb perfusion in normal subjects and patients with peripheral artery disease (PAD). BACKGROUND: US-induced cavitation of microbubble contrast agents augments tissue perfusion by convective shear and secondary purinergic signaling that mediates release of endogenous vasodilators. METHODS: In mice, unilateral exposure of the proximal hindlimb to therapeutic US (1.3 MHz, mechanical index 1.3) was performed for 10 min after intravenous injection of lipid microbubbles. US varied according to line density (17, 37, 65 lines) and pulse duration. Microvascular perfusion was evaluated by US perfusion imaging, and in vivo adenosine triphosphate (ATP) release was assessed using in vivo optical imaging. Optimal parameters were then used in healthy volunteers and patients with PAD where calf US alone or in combination with intravenous microbubble contrast infusion was performed for 10 min. RESULTS: In mice, flow was augmented in the US-exposed limb for all acoustic conditions. Only at the lowest line density was there a stepwise increase in perfusion for longer (40-cycle) versus shorter (5-cycle) pulse duration. For higher line densities, blood flow consistently increased by 3-fold to 4-fold in the US-exposed limb irrespective of pulse duration. High line density and long pulse duration resulted in the greatest release of ATP in the cavitation zone. Application of these optimized conditions in humans together with intravenous contrast increased calf muscle blood flow by >2-fold in both healthy subjects and patients with PAD, whereas US alone had no effect. CONCLUSIONS: US of microbubbles when using optimized acoustic environments can increase perfusion in limb skeletal muscle, raising the possibility of a therapy for patients with PAD. (Augmentation of Limb Perfusion With Contrast Ultrasound; NCT03195556).
Subject(s)
Contrast Media/administration & dosage , Muscle, Skeletal/blood supply , Peripheral Arterial Disease/therapy , Ultrasonic Therapy , Aged , Animals , Blood Flow Velocity , Disease Models, Animal , Female , Hindlimb , Humans , Injections, Intravenous , Leg , Male , Mice , Mice, Inbred C57BL , Microbubbles , Middle Aged , Peripheral Arterial Disease/diagnostic imaging , Peripheral Arterial Disease/physiopathology , Regional Blood Flow , Treatment OutcomeABSTRACT
BACKGROUND: Ultrasound-mediated cavitation of microbubble contrast agents produces high intravascular shear. We hypothesized that microbubble cavitation increases myocardial microvascular perfusion through shear-dependent purinergic pathways downstream from ATP release that is immediate and sustained through cellular ATP channels such as Pannexin-1. METHODS: Quantitative myocardial contrast echocardiography perfusion imaging and in vivo optical imaging of ATP was performed in wild-type and Pannexin-1-deficient (Panx1-/-) mice before and 5 and 30 minutes after 10 minutes of ultrasound-mediated (1.3 MHz, mechanical index 1.3) myocardial microbubble cavitation. Flow augmentation in a preclinical model closer to humans was evaluated in rhesus macaques undergoing myocardial contrast echocardiography perfusion imaging after high-power cavitation in the apical four-chamber plane for 10 minutes. RESULTS: Microbubble cavitation in wild-type mice (n = 7) increased myocardial perfusion by 64% ± 25% at 5 minutes and 95% ± 55% at 30 minutes compared with baseline (P < .05). In Panx1-/- mice (n = 5), perfusion increased by 28% ± 26% at 5 minutes (P = .04) but returned to baseline at 30 minutes. Myocardial ATP signal in wild-type (n = 7) mice undergoing cavitation compared with sham-treated controls (n = 3) was 450-fold higher at 5 minutes and 90-fold higher at 30 minutes after cavitation (P < .001). The ATP signal in Panx1-/- mice (n = 4) was consistently 10-fold lower than that in wild-type mice and was similar to sham controls at 30 minutes. In macaques (n = 8), myocardial perfusion increased twofold in the cavitation-exposed four-chamber plane, similar in degree to that produced by adenosine, but did not increase in the control two-chamber plane. CONCLUSIONS: Cavitation of microbubbles in the myocardial microcirculation produces an immediate release of ATP, likely from cell microporation, as well as sustained release, which is channel dependent and responsible for persistent flow augmentation. These findings provide mechanistic insight by which cavitation improves perfusion and reduces infarct size in patients with myocardial infarction.
Subject(s)
Contrast Media , Microbubbles , Animals , Connexins , Macaca mulatta , Mice , Mice, Inbred C57BL , Myocardium , Nerve Tissue Proteins , UltrasonographyABSTRACT
The ability to accurately evaluate skeletal muscle microvascular blood flow has broad clinical applications for understanding the regulation of skeletal muscle perfusion in health and disease states. Contrast-enhanced ultrasound (CEU) perfusion imaging, a technique originally developed to evaluate myocardial perfusion, is one of many techniques that have been applied to evaluate skeletal muscle perfusion. Among the advantages of CEU perfusion imaging of skeletal muscle is that it is rapid, safe and performed with equipment already present in most vascular medicine laboratories. The aim of this review is to discuss the use of CEU perfusion imaging in skeletal muscle. This article provides details of the protocols for CEU imaging in skeletal muscle, including two predominant methods for bolus and continuous infusion destruction-replenishment techniques. The importance of stress perfusion imaging will be highlighted, including a discussion of the methods used to produce hyperemic skeletal muscle blood flow. A broad overview of the disease states that have been studied in humans using CEU perfusion imaging of skeletal muscle will be presented including: (1) peripheral arterial disease; (2) sickle cell disease; (3) diabetes; and (4) heart failure. Finally, future applications of CEU imaging in skeletal muscle including therapeutic CEU imaging will be discussed along with technological developments needed to advance the field.
ABSTRACT
OBJECTIVES: This study evaluated whether lipoprotein apheresis produces immediate changes in resting perfusion in subjects with severe hypercholesterolemia, and whether there is a difference in the response between peripheral and coronary microcirculations. BACKGROUND: Lipoprotein apheresis is used in patients with severe hypercholesterolemia to reduce plasma levels of low-density lipoprotein cholesterol. METHODS: Quantitative contrast-enhanced ultrasound perfusion imaging of the myocardium at rest and skeletal muscle at rest and during calibrated contractile exercise was performed before and immediately after lipoprotein apheresis in 8 subjects with severe hypercholesterolemia, 7 of whom had a diagnosis of familial hypercholesterolemia. Myocardial perfusion imaging was also performed in 14 normal control subjects. Changes in myocardial work and left ventricular function were assessed by echocardiography. Ex vivo ovine coronary and femoral artery ring tension assays were assessed in the presence of pre- and post-apheresis plasma. RESULTS: Apheresis acutely decreased low-density lipoprotein cholesterol (234.9 ± 103.2 mg/dl vs. 67.1 ± 49.5 mg/dl; p < 0.01) and oxidized phospholipid on apolipoprotein B-100 (60.2 ± 55.2 nmol/l vs. 47.0 ± 24.5 nmol/l; p = 0.01), and acutely increased resting myocardial perfusion (55.1 [95% confidence interval: 77.2 to 73.1] IU/s vs. 135 [95% confidence interval: 81.2 to 189.6] IU/s; p = 0.01), without changes in myocardial work. Myocardial longitudinal strain improved in those subjects with reduced pre-apheresis function. Skeletal muscle perfusion at rest and during contractile exercise was unchanged by apheresis. Acetylcholine-mediated dilation of ex vivo ovine coronary but not femoral arteries was impaired in pre-apheresis plasma and was completely reversed in post-apheresis plasma. CONCLUSIONS: Lipoprotein apheresis produces an immediate improvement in coronary microvascular function, which increases myocardial perfusion and normalizes endothelial-dependent vasodilation. These changes are not observed in the periphery. (Acute Microvascular Changes With LDL Apheresis; NCT02388633).
Subject(s)
Blood Component Removal , Cholesterol, LDL/blood , Coronary Circulation , Coronary Disease/physiopathology , Hypercholesterolemia/therapy , Microcirculation , Muscle, Skeletal/blood supply , Peripheral Arterial Disease/physiopathology , Aged , Animals , Biomarkers/blood , Blood Component Removal/adverse effects , Case-Control Studies , Coronary Disease/blood , Coronary Disease/diagnostic imaging , Coronary Disease/etiology , Down-Regulation , Female , Humans , Hypercholesterolemia/blood , Hypercholesterolemia/complications , Hypercholesterolemia/diagnosis , Male , Middle Aged , Peripheral Arterial Disease/blood , Peripheral Arterial Disease/diagnostic imaging , Peripheral Arterial Disease/etiology , Recovery of Function , Severity of Illness Index , Sheep, Domestic , Time Factors , Treatment OutcomeABSTRACT
BACKGROUND: In patients with peripheral artery disease (PAD), the severity of symptoms correlates poorly with ankle-brachial index (ABI). The aim of this study was to test the hypothesis that limb perfusion assessed using contrast-enhanced ultrasound (CEU) during contractile exercise varies according to functional class in patients with PAD, particularly those with ABIs in the 0.4 to 0.6 range whose symptoms vary widely. METHODS: Bilateral quantitative CEU perfusion imaging of the calf was performed in normal control subjects (n = 10) and patients with PAD who had at least one limb with a moderately reduced ABI (0.4-0.6; n = 17). Imaging was performed at rest and immediately after 30 sec of modest periodic (0.3-Hz) plantar flexion (10 W). RESULTS: In patients with PAD, Rutherford symptom classification for each limb varied widely, including in limbs with ABIs of 0.4 to 0.6 (n = 6 with mild or no symptoms, n = 14 with moderate to severe symptoms). CEU perfusion imaging parameters at rest were similar between control subjects and patients with PAD irrespective of ABI. In normal control subjects, limb flow increased on average by > 20-fold after only 30 sec of moderate exercise. In patients with PAD, muscle exercise perfusion for all limbs was reduced compared with control subjects and decreased according to the severity of ABI reduction, primarily from reduced microvascular flux rate. Even limbs with ABIs > 0.9 in patients with PAD had lower exercise perfusion than in control subjects (P = .03). In subjects with PAD, exercise perfusion was lower in those with moderate to severe versus mild symptoms when analyzed for all limbs (median, 30 IU/sec [interquartile range (IQR), 21-52 IU/sec] vs 84 IU/sec [IQR, 36-177 IU/sec]; P = .01) and limbs with ABIs of 0.4 to 0.6 (median, 26 IU/sec [IQR, 14-41 IU/sec] vs 54 IU/sec [IQR, 31-105 IU/sec]; P = .05). CONCLUSIONS: In patients with PAD, CEU exercise perfusion imaging detects differences in limb muscle perfusion that are likely to be responsible for differences in symptom severity and can detect the flow abnormalities from microvascular dysfunction even in limbs with normal ABIs.
Subject(s)
Ankle Brachial Index/methods , Exercise/physiology , Leg/blood supply , Perfusion Imaging/methods , Peripheral Arterial Disease/diagnosis , Regional Blood Flow/physiology , Ultrasonography/methods , Aged , Exercise Test/methods , Female , Humans , Male , Middle Aged , Peripheral Arterial Disease/physiopathology , Severity of Illness IndexABSTRACT
BACKGROUND: Microvascular dysfunction (MVD) is a potential cause of chest pain in younger individuals. The authors hypothesized that nonelderly patients referred for computed tomographic angiography (CTA) but without significant stenosis would have a high prevalence of MVD by myocardial contrast echocardiography (MCE). Secondary aims were to test whether the presence of nonobstructive coronary artery disease (CAD) or reduced brachial flow-mediated dilation (FMD) predicted MVD. METHODS: Subjects ≤60 years of age undergoing CTA were recruited if they had either no evidence of coronary plaque or evidence of mild CAD (<50% stenosis) and at least one high-risk plaque feature. Subjects underwent quantitative perfusion imaging using MCE at rest and during regadenoson vasodilator stress. MVD was defined as global or segmental delay of microvascular refill (≥2 sec) during regadenoson. FMD of the brachial artery was also performed. RESULTS: Of the 29 patients in whom MCE could be performed, 12 (41%) had MVD. These subjects, compared with those with normal microvascular function, had lower hyperemic perfusion (mean, 236 ± 68 vs 354 ± 161 intensity units/sec; P = .02) and microvascular flux rate (mean, 1.6 ± 0.4 vs 2.5 ± 0.9 sec-1; P = .002) on quantitative MCE. The degree of FMD was not significantly different in those with or without MVD (mean, 11 ± 4% vs 9 ± 4%; P = .32), and there was a poor correlation between results on stress MCE and FMD. Only eight of the 29 subjects were classified as having nonobstructive CAD. There were no groupwise differences in the prevalence of MVD function in those with versus without CAD (43% vs 38% for negative and positive findings on CTA, respectively, P = .79). CONCLUSIONS: MVD is a common finding in the nonelderly population referred for CTA for evaluation of possible CAD but without obstructive stenosis. Neither the presence of noncritical atherosclerotic disease nor abnormal FMD increases the likelihood for detecting MVD in this population.
Subject(s)
Computed Tomography Angiography , Coronary Artery Disease/diagnostic imaging , Echocardiography , Microvascular Angina/diagnostic imaging , Adult , Brachial Artery/diagnostic imaging , Chest Pain/diagnostic imaging , Female , Humans , Iohexol , Male , Middle Aged , Oregon , Prospective Studies , Purines , PyrazolesABSTRACT
The aim of this study was to evaluate a panel of endothelium-targeted microbubble (MB) ultrasound contrast agents bearing small peptide ligands as a human-ready approach for molecular imaging of markers of high-risk atherosclerotic plaque. Small peptide ligands with established affinity for human P-selectin, VCAM-1, LOX-1 and von Willebrand factor (VWF) were conjugated to the surface of lipid-stabilized MBs. Contrast-enhanced ultrasound (CEUS) molecular imaging of the thoracic aorta was performed in wild-type and gene-targeted mice with advanced atherosclerosis (DKO). Histology was performed on carotid endarterectomy samples from patients undergoing surgery for unstable atherosclerosis to assess target expression in humans. In DKO mice, CEUS signal for all four targeted MBs was significantly higher than that for control MBs, and was three to sevenfold higher than in wild-type mice, with the highest signal achieved for VCAM-1 and VWF. All molecular targets were present on the patient plaque surface but expression was greatest for VCAM-1 and VWF. We conclude that ultrasound contrast agents bearing small peptide ligands feasible for human use can be targeted against endothelial cell adhesion molecules for inflammatory cells and platelets for imaging advanced atherosclerotic disease.
Subject(s)
Atherosclerosis/diagnostic imaging , Endothelium, Vascular/diagnostic imaging , Inflammation/diagnostic imaging , Oxidative Stress , Ultrasonography/methods , Animals , Atherosclerosis/physiopathology , Contrast Media , Disease Models, Animal , Endothelium, Vascular/physiopathology , Image Enhancement/methods , Inflammation/physiopathology , Ligands , Mice , Mice, Inbred C57BL , Microbubbles , Molecular Imaging/methods , PeptidesABSTRACT
BACKGROUND: Ultrasound molecular imaging was used to evaluate the therapeutic effects of antioxidant therapy with EUK-207, which has superoxide dismutase and catalase activities, on suppressing high-risk atherosclerotic features. METHODS: Mice with age-dependent atherosclerosis produced by deletion of the low-density lipoprotein receptor and Apobec-1 were studied at 20 and 40 weeks of age. EUK-207 or vehicle was administered for the preceding 8 weeks. Therapy for 28 weeks was also studied for 40-week-old mice. Ultrasound molecular imaging of the thoracic aorta was performed with contrast agents targeted to endothelial P-selectin, von Willebrand factor A1-domain, and platelet glycoprotein Ibα or control agent. Aortic plaque area and macrophage content were assessed by histology. RESULTS: In 20-week-old double-knockout mice, EUK-207 compared with sham therapy produced only nonsignificant trends for reduction in molecular imaging signal for endothelial P-selectin, von Willebrand factor A1-domain, and platelet adhesion. At 40 weeks, EUK-207 given for 8 or 28 weeks significantly (P < .05) reduced signal for all three endothelial-associated events essentially to background levels, with the exception of glycoprotein Ibα signal after 8 weeks (P = .06). On aortic histology, EUK-207 therapy for 8 weeks did not affect plaque area or macrophage content at either age. However, EUK-207 for 28 weeks almost completely suppressed plaque development (350 ± 258 vs 4 ± 6 × 103 µm2, P = .014) and macrophage content (136 ± 103 vs 3 ± 2 × 103 µm2, P = .002) compared with control mice at 40 weeks. CONCLUSIONS: Molecular imaging can be used to assess vascular responses to antioxidants and has demonstrated that certain antioxidants reduce vascular endothelial activation and platelet adhesion, but reductions in plaque size and macrophage content occurs only with long-duration therapy that is started early.
Subject(s)
Antioxidants/therapeutic use , Aorta, Thoracic/diagnostic imaging , Aortic Diseases/drug therapy , Atherosclerosis/drug therapy , Contrast Media/pharmacology , Molecular Imaging/methods , Ultrasonography/methods , Animals , Aortic Diseases/diagnosis , Atherosclerosis/diagnosis , Disease Models, Animal , Mice , Mice, Inbred C57BL , Mice, Knockout , Treatment OutcomeABSTRACT
BACKGROUND: Contrast-enhanced ultrasound (CEU) limb perfusion imaging is a promising approach for evaluating peripheral artery disease (PAD). However, low signal enhancement in skeletal muscle has necessitated high-power intermittent imaging algorithms, which are not clinically feasible. We hypothesized that CEU using a combination of intermediate power and a contrast agent resistant to inertial cavitation would allow real-time limb stress perfusion imaging. METHODS: In normal volunteers, CEU of the calf skeletal muscle was performed on separate days with Sonazoid, Optison, or Definity. Progressive reduction in the ultrasound pulsing interval was used to assess the balance between signal enhancement and agent destruction at escalating mechanical indices (MI, 0.1-0.4). Real-time perfusion imaging at MI 0.1-0.4 using postdestructive replenishment kinetics was performed at rest and during 25 W plantar flexion contractile exercise. RESULTS: For Optison, limb perfusion imaging was unreliable at rest due to very low signal enhancement generated at all MIs and was possible during exercise-induced hyperemia only at MI 0.1 due to agent destruction at higher MIs. For Definity, signal intensity progressively increased with MI but was offset by microbubble destruction, which resulted in modest signal enhancement during CEU perfusion imaging and distortion of replenishment curves at MI ≥ 0.2. For Sonazoid, there strong signal enhancement at MI ≥ 0.2, with little destruction detected only at MI 0.4. Accordingly, high signal intensity and nondistorted perfusion imaging was possible at MI 0.2-0.3 and detected an 8.0- ± 5.7-fold flow reserve. CONCLUSIONS: Rest-stress limb perfusion imaging in humans with real-time CEU, which requires only seconds to perform, is possible using microbubbles with viscoelastic properties that produce strong nonlinear signal generation without destruction at intermediate acoustic pressures.