Influence of Atherosclerotic Risk Factors on the Effectiveness of Therapeutic Ultrasound Cavitation for Flow Augmentation.

BACKGROUND: Shear created by inertial cavitation of microbubbles by ultrasound augments limb and myocardial perfusion and can reverse tissue ischemia. Our aim was to determine whether this therapeutic bioeffect is attenuated by atherosclerotic risk factors that are known to impair shear-mediated vasodilation and adversely affect microvascular reactivity. METHODS: In mice, lipid-stabilized decafluorobutane microbubbles (2 × 108) were administered intravenously while exposing a proximal hind limb to ultrasound (1.3 MHz, 1.3 mechanical index, pulsing interval 5 seconds) for 10 minutes. Murine strains included wild-type mice and severely hyperlipidemic mice at 15, 35, or 52 weeks of age as a model of aging and elevated cholesterol, and obese db/db mice (≈15 weeks) with severe insulin resistance. Quantitative contrast-enhanced ultrasound perfusion imaging was performed to assess microvascular perfusion in the control and ultrasound-exposed limb. An in situ electrochemical probe and in vivo biophotonic imaging were used to assess limb nitric oxide (NO) and adenosine triphosphosphate concentrations, respectively. RESULTS: Microvascular perfusion was significantly increased by several fold in the cavitation-exposed limb versus control limb for all murine strains and ages (P < .001). In wild-type and hyperlipidemic mice, hyperemia from cavitation was attenuated in the 2 older age groups (P < .01). In young mice (15 weeks), perfusion in cavitation-exposed muscle was less in both the hyperlipidemic mice and the obese db/db mice compared with corresponding wild-type mice. Using young hyperlipidemic mice as a model for flow impairment, limb NO production after cavitation was reduced but adenosine triphosphosphate production was unaltered when compared with age-matched wild-type mice. CONCLUSIONS: In mice, ultrasound cavitation of microbubbles increases limb perfusion by several fold even in the presence of traditional atherosclerotic risk factors. However, older age, hyperlipidemia, and insulin resistance modestly attenuate the degree of flow augmentation, which could impact the degree of flow response in current clinical trials in patients with critical limb ischemia.

Increased Susceptibility for Adverse Reactions to Ultrasound Enhancing Agents in Sickle Cell Disease.

BACKGROUND: Pain-related adverse events (AEs) to ultrasound enhancing agents (UEAs) have been reported in patients with sickle cell disease (SCD). The aims of this study were to characterize the scope of these AEs in the SCD population and to investigate potential mechanisms on the basis of pathways involved in SCD vaso-occlusive crisis (VOC) and pain. METHODS: The prevalence and classification of AEs were analyzed from two clinical trials in which high-dose Definity infusions were used in patients with SCD (n = 55) or matched control subjects (n = 43) to study muscle or myocardial microvascular perfusion. Because complement (C') activation can trigger VOC in SCD, C' activation and surface adhesion of C' proteins on lipid UEAs were studied in vitro. C'-mediated UEA attachment to bone marrow immune cells was assessed using flow cytometry in a murine SCD model (Townes mice). Blood from patients receiving Definity was obtained to measure specific lysophospholipid metabolites of lipids in Definity thought to mediate SCD pain. RESULTS: Moderate or greater AEs, all of which were nociceptive (back or bone pain), occurred in one control subject and nine SCD subjects (2% vs 16%, P = .02). Patients with SCD who had AEs tended to have more severe manifestations of SCD. Three of the subjects with SCD had previously received Definity without complications. In patients with SCD, four AEs were classified as severe in intensity and as serious AEs on the basis of need for medical intervention. AEs were described to be similar to SCD-related pain, but there was no evidence for VOC, hemolysis, hypotension, or hypoxemia. At baseline, markers of C' activation were greater in patients with SCD than control subjects. However, after administration of lipid UEAs, SCD and control subjects were similar with regard to C' activation response, anaphylatoxin production, bone marrow microbubble retention, and production of lysophospholipids. There was a trend toward increased deposition of C3b and C3bi on lipid UEAs exposed to serum from patients with SCD. CONCLUSIONS: Patients with SCD are particularly susceptible to nociceptive AEs when given Definity at high doses. The mechanism for these AEs remains unclear but most are not related to the triggering of classic VOC.

Myocardial contrast echocardiography assessment of perfusion abnormalities in hypertrophic cardiomyopathy.

BACKGROUND: Perfusion defects during stress can occur in hypertrophic cardiomyopathy (HCM) from either structural or functional abnormalities of the coronary microcirculation. In this study, vasodilator stress myocardial contrast echocardiography (MCE) was used to quantify and spatially characterize hyperemic myocardial blood flow (MBF) deficits in HCM. METHODS: Regadenoson stress MCE was performed in patients with septal-variant HCM (n = 17) and healthy control subjects (n = 15). The presence and spatial distribution (transmural diffuse, patchy, subendocardial) of perfusion defects was determined by semiquantitative analysis. Kinetic analysis of time-intensity data was used to quantify MBF, microvascular flux rate (ß), and microvascular blood volume. In patients undergoing septal myectomy (n = 3), MCE was repeated > 1 years after surgery.  RESULTS: In HCM subjects, perfusion defects during stress occurred in the septum in 80%, and in non-hypertrophied regions in 40%. The majority of septal defects (83%) were patchy or subendocardial, while 67% of non-hypertrophied defects were transmural and diffuse. On quantitative analysis, hyperemic MBF was approximately 50% lower (p < 0.001) in the hypertrophied and non-hypertrophied regions of those with HCM compared to controls, largely based on an inability to augment ß, although hypertrophic regions also had blood volume deficits. There was no correlation between hyperemic MBF and either percent fibrosis on magnetic resonance imaging or outflow gradient, yet those with higher degrees of fibrosis (≥ 5%) or severe gradients all had low septal MBF during regadenoson. Substantial improvement in hyperemic MBF was observed in two of the three subjects undergoing myectomy, both of whom had severe pre-surgical outflow gradients at rest. CONCLUSION: Perfusion defects on vasodilator MCE are common in HCM, particularly in those with extensive fibrosis, but have a different spatial pattern for the hypertrophied and non-hypertrophied segments, likely reflecting different contributions of functional and structural abnormalities. Improvement in hyperemic perfusion is possible in those undergoing septal myectomy to relieve obstruction.  TRIAL REGISTRATION: ClinicalTrials.gov NCT02560467.

Contrast Ultrasound Assessment of Skeletal Muscle Recruitable Perfusion after Permanent Left Ventricular Assist Device Implantation: Implications for Functional Recovery.

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.

Plasma Lipidomic Patterns in Patients with Symptomatic Coronary Microvascular Dysfunction.

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.

Limb Perfusion During Exercise Assessed by Contrast Ultrasound Varies According to Symptom Severity in Patients with Peripheral Artery Disease.

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.

Coronary Microvascular Dysfunction by Myocardial Contrast Echocardiography in Nonelderly Patients Referred for Computed Tomographic Coronary Angiography.

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.

Lipoprotein Apheresis Acutely Reverses Coronary Microvascular Dysfunction in Patients With Severe Hypercholesterolemia.

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).

Ultrasound Molecular Imaging of Atherosclerosis Using Small-Peptide Targeting Ligands Against Endothelial Markers of Inflammation and Oxidative Stress.

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.

Exercise versus vasodilator stress limb perfusion imaging for the assessment of peripheral artery disease.

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 (

Augmentation of Muscle Blood Flow by Ultrasound Cavitation Is Mediated by ATP and Purinergic Signaling.

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.

Rest-Stress Limb Perfusion Imaging in Humans with Contrast Ultrasound Using Intermediate-Power Imaging and Microbubbles Resistant to Inertial Cavitation.

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.

Functional adaptations of the coronary microcirculation to anaemia in fetal sheep.

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.

Influence of DNA-Microbubble Coupling on Contrast Ultrasound-Mediated Gene Transfection in Muscle and Liver.

BACKGROUND: Contrast ultrasound-mediated gene delivery (CUMGD) is a promising approach for enhancing gene therapy that relies on microbubble (MB) cavitation to augment complementary deoxyribonucleic acid (cDNA) transfection. The aims of this study were to determine optimal conditions for charge-coupling cDNA to MBs and to evaluate the advantages of surface loading for gene transfection in muscle and liver. METHODS: Charge coupling of fluorescently labeled cDNA to either neutral MBs (MBN) or cationic MBs (MB+) in low- to high-ionic conditions (0.3%-1.8% NaCl) was assessed by flow cytometry. MB aggregation from cDNA coupling was determined by electrozone sensing. Tissue transfection of luciferase in murine hindlimb skeletal muscle and liver was made by CUMGD with MBN or MB+ combined with subsaturated, saturated, or supersaturated cDNA concentrations (2.5, 50, and 200 µg/10(8) MBs). RESULTS: Charge-coupling of cDNA was detected for MB+ but not MBN. Coupling occurred over almost the entire range of ionic conditions, with a peak at 1.2% NaCl, although electrostatic interference occurred at >1.5% NaCl. DNA-mediated aggregation of MB+ was observed at ≤0.6% NaCl but did not reduce the ability to produce inertial cavitation. Transfection with CUMGD in muscle and liver was low for both MBs at subsaturation concentrations. In muscle, higher cDNA concentrations produced a 10-fold higher degree of transfection with MB+, which was approximately fivefold higher (P < .05) than that for MBN. There was no effect of DNA supersaturation. The same pattern was seen for liver except that supersaturation further increased transfection with MBN equal to that of MB+. CONCLUSIONS: Efficient charge-coupling of cDNA to MB+ but not MBN occurs over a relatively wide range of ionic conditions without aggregation. Transfection with CUMGD is much more efficient with charge-coupling of cDNA to MBs and is not affected by supersaturation except in the liver, which is specialized for macromolecular and cDNA uptake.

Echocardiographic Ischemic Memory Imaging Through Complement-Mediated Vascular Adhesion of Phosphatidylserine-Containing Microbubbles.

OBJECTIVES: This study hypothesized that microvascular retention of phosphatidylserine-containing microbubbles (MB-PS) would allow detection of recent but resolved myocardial ischemia with myocardial contrast echocardiographic (MCE) molecular imaging. BACKGROUND: Techniques for ischemic memory imaging which can detect and spatially assess resolved myocardial ischemia are being developed for rapid evaluation of patients with chest pain. METHODS: MCE molecular imaging with MB-PS was performed 1.5 h, 3.0 h, and 6.0 h after brief (10 min) myocardial ischemia in mice; data were compared to selectin-targeted microbubbles. MCE molecular imaging with Sonazoid (GE Healthcare, Amersham, United Kingdom), a commercially produced phosphatidylserine (PS) - containing agent, was performed in separate mice at 1.5 h and 3.0 h after ischemia-reperfusion; and in dogs undergoing 135 min of ischemia and 60 min of reflow as well as in closed-chest nonischemic control dogs. The mechanism for MB-PS attachment was assessed by intravital microscopy of post-ischemic muscle and by flow cytometry analysis of cell-MB interactions. RESULTS: In mice undergoing ischemia-reperfusion without infarction, signal enhancement in the risk area for MB-PS and p-selectin glycoprotein ligand-1-targeted microbubbles was similar at reflow times of 1.5 h (23.3 ± 7.3 IU vs. 30.7 ± 4.1 IU), 3.0 h (42.2 ± 6.2 IU vs. 33.9 ± 7.4 IU), and 6.0 h (24.1 ± 4.3 IU vs. 25.5 ± 4.7 IU). For both agents, signal in the risk area was significantly (p < 0.05) higher than remote region at all reflow times. Sonazoid also produced strong risk area enhancement at 1.5 h (34.7 ± 5.0 IU) and 3.0 h (52.5 ± 4.5 IU) which was approximately 3-fold greater than in the control region, and which correlated spatially with the microsphere-derived risk area. In dogs, Sonazoid signal in the risk area was >5-fold higher than in closed-chest control myocardium (42.2 ± 8.1 IU vs. 7.9 ± 3.3 IU; p < 0.001). Mechanistic studies indicated that MB-PS attached directly to venular endothelium and adherent leukocytes which was dependent on serum complement components C1q and C3. CONCLUSIONS: Ischemic memory imaging with MCE is possible using MB-PS which may obviate the need for ligand-directed targeting.

Temporal Changes in Skeletal Muscle Capillary Responses and Endothelial-Derived Vasodilators in Obesity-Related Insulin Resistance.

The inability of insulin to increase skeletal muscle capillary blood volume (CBV) reduces glucose uptake in insulin resistance (IR). We hypothesized that abnormalities in endothelial-derived vasodilator pathways are temporally associated with the development of IR and an impaired ability to increase skeletal muscle CBV. A comprehensive metabolic and vascular screening assessment was performed on 10 adult rhesus macaques at baseline and every 4-6 months for 2 years after starting a high-fat diet supplemented with fructose. Diet changes resulted in an 80% increase in truncal fat by 4 months. Hyperinsulinemia and decreased glucose utilization were observed from 4 to 18 months. At 24 months, pancreatic secretory function and the glucose utilization rate declined. CBV at rest and during an intravenous glucose tolerance test demonstrated a sustained increase from 4 to 18 months and then abruptly fell at 24 months. Nitric oxide bioavailability progressively decreased over 2 years. Conversely, endothelial-derived vasodilators progressively increased over 18 months and then abruptly decreased at 24 months in concert with the CBV. The increase in basal and glucose-mediated CBV early in IR may represent a compensatory response through endothelial-derived vasodilator pathways. The inability to sustain a vascular compensatory response limits glucose-mediated increases in CBV, which correlates with the severity of IR.

Quantification of residual limb skeletal muscle perfusion with contrast-enhanced ultrasound during application of a focal junctional tourniquet.

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.

Abnormal Regulation of Microvascular Tone in a Murine Model of Sickle Cell Disease Assessed by Contrast Ultrasound.

BACKGROUND: Microvascular dysregulation, abnormal rheology, and vaso-occlusive events play a role in the pathophysiology of sickle cell disease (SCD). The aim of this study was to test the hypothesis that abnormalities in skeletal muscle perfusion in a murine model of SCD could be parametrically assessed by quantitative contrast-enhanced ultrasound perfusion imaging. METHODS: A murine model of moderate SCD without anemia produced by homozygous ß-globin deletion replaced by human ßs-globin transgene (NY1DD-/-; n = 18), heterozygous transgene replacement (NY1DD+/-; n = 19), and C57Bl/6 control mice (n = 14) was studied. Quantitative contrast-enhanced ultrasound of the proximal hindlimb skeletal muscle was performed at rest and during contractile exercise (2 Hz). Time-intensity data were analyzed to measure microvascular blood volume (MBV), microvascular blood transit rate (ß), and microvascular blood flow. Erythrocyte deformability was measured by elongation at various rotational shears. RESULTS: At rest, muscle MBV was similar between strains, whereas ß was significantly (P = .0015, analysis of variance) reduced to a similar degree in NY1DD-/- and NY1DD+/- compared with wild-type mice (0.24 ± 0.10, 0.16 ± 0.07, and 0.34 ± 0.14 sec(-1), respectively), resulting in a reduction in microvascular blood flow. During contractile exercise, there were no groupwise differences in ß (1.43 ± 0.67, 1.09 ± 0.42, and 1.36 ± 0.49 sec(-1) for NY1DD-/-, NY1DD+/-, and wild-type mice, respectively) or in microvascular blood flow or MBV. Erythrocyte deformability at high shear stress (≥5 Pa) was mildly reduced in both transgenic groups, although it was not correlated with blood flow or ß. CONCLUSIONS: Contrast-enhanced ultrasound in skeletal muscle revealed a lower microvascular blood transit rate in the NY1DD model of SCD and sickle trait but no alterations in MBV. The abnormality in microvascular blood transit rate was likely due to vasomotor dysfunction, because it was abrogated by contractile exercise and at rest was only weakly related to erythrocyte deformability.