Therapeutic Ultrasound Increases Myocardial Blood Flow in Ischemic Myocardium and Cardiac Endothelial Cells: Results of In Vivo and In Vitro Experiments.

BACKGROUND: Therapeutic ultrasound can reduce infarct size in a model of coronary thrombosis even when sonothrombolysis is ineffective. The aim of this study was to test the hypothesis that ultrasound-induced cardioprotection is mediated by molecules released from the vascular endothelium that increase myocardial blood flow (MBF) and also have direct tissue-salvaging effects. METHODS: In vivo and in vitro experiments were performed using a 1.05-MHz transducer. For the in vivo experiments 10 control and 10 ultrasound-treated dogs undergoing occlusion of the left anterior descending coronary artery were studied. MBF was measured using myocardial contrast echocardiography. For the in vitro experiments, primary mouse cardiac endothelial cells were exposed to ultrasound at baseline or following oxygen-glucose deprivation and endothelial nitric oxide synthase phosphorylation as well as adenosine and the eicosanoids epoxyeicosatrienoic acids, dihydroxyeicosatrienoic acids, and hydroxyl-eicosatetraenoic acids were measured. RESULTS: In vivo, ultrasound treatment caused higher MBF (20 ± 10 vs 10 ± 8, P = .03) and higher wall thickening (3 ± 3% vs 1 ± 0.4%, P = .01) in the collateral-derived border zone compared with control. Epicardial MBF in the left anterior descending coronary artery bed also tended to be higher (17 ± 17 vs 5 ± 4, P = .05) in ultrasound-treated versus control animals; however, endocardial MBF in this region was similar to that in controls (13 ± 14 vs 14 ± 7). In vitro, phosphorylated endothelial nitric oxide synthase and adenosine increased (by 129 ± 11% and 286 ± 63%, respectively, P < .01) with ultrasound compared with unstimulated cells. Similar results were obtained with epoxyeicosatrienoic acids. After oxygen-glucose deprivation, phosphorylated endothelial nitric oxide synthase decreased and was restored with application of ultrasound. Similar changes were noted with epoxyeicosatrienoic acids. Cell viability decreased with oxygen-glucose deprivation and returned to near baseline with ultrasound. CONCLUSIONS: Ultrasound increases MBF in ischemic tissue in vivo. This effect is likely mediated by the release of a plethora of coronary vasodilators during ultrasound treatment that also have direct tissue-salvaging effects. Therapeutic ultrasound, therefore, has potential for treatment of acute and chronic myocardial ischemia independent of its effect on thrombolysis.

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.

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.

Augmentation of limb perfusion and reversal of tissue ischemia produced by ultrasound-mediated microbubble cavitation.

BACKGROUND: Ultrasound can increase tissue blood flow, in part, through the intravascular shear produced by oscillatory pressure fluctuations. We hypothesized that ultrasound-mediated increases in perfusion can be augmented by microbubble contrast agents that undergo ultrasound-mediated cavitation and sought to characterize the biological mediators. METHODS AND RESULTS: Contrast ultrasound perfusion imaging of hindlimb skeletal muscle and femoral artery diameter measurement were performed in nonischemic mice after unilateral 10-minute exposure to intermittent ultrasound alone (mechanical index, 0.6 or 1.3) or ultrasound with lipid microbubbles (2×10(8) IV). Studies were also performed after inhibiting shear- or pressure-dependent vasodilator pathways, and in mice with hindlimb ischemia. Ultrasound alone produced a 2-fold increase (P<0.05) in muscle perfusion regardless of ultrasound power. Ultrasound-mediated augmentation in flow was greater with microbubbles (3- and 10-fold higher than control for mechanical index 0.6 and 1.3, respectively; P<0.05), as was femoral artery dilation. Inhibition of endothelial nitric oxide synthase attenuated flow augmentation produced by ultrasound and microbubbles by 70% (P<0.01), whereas inhibition of adenosine-A2a receptors and epoxyeicosatrienoic acids had minimal effect. Limb nitric oxide production and muscle phospho-endothelial nitric oxide synthase increased in a stepwise fashion by ultrasound and ultrasound with microbubbles. In mice with unilateral hindlimb ischemia (40%-50% reduction in flow), ultrasound (mechanical index, 1.3) with microbubbles increased perfusion by 2-fold to a degree that was greater than the control nonischemic limb. CONCLUSIONS: Increases in muscle blood flow during high-power ultrasound are markedly amplified by the intravascular presence of microbubbles and can reverse tissue ischemia. These effects are most likely mediated by cavitation-related increases in shear and activation of endothelial nitric oxide synthase.

Real-time contrast ultrasound muscle perfusion imaging with intermediate-power imaging coupled with acoustically durable microbubbles.

BACKGROUND: There is growing interest in limb contrast-enhanced ultrasound (CEU) perfusion imaging for the evaluation of peripheral artery disease. Because of low resting microvascular blood flow in skeletal muscle, signal enhancement during limb CEU is prohibitively low for real-time imaging. The aim of this study was to test the hypothesis that this obstacle can be overcome by intermediate- rather than low-power CEU when performed with an acoustically resilient microbubble agent. METHODS: Viscoelastic properties of Definity and Sonazoid were assessed by measuring bulk modulus during incremental increases in ambient pressure to 200 mm Hg. Comparison of in vivo microbubble destruction and signal enhancement at a mechanical index (MI) of 0.1 to 0.4 was performed by sequential reduction in pulsing interval from 10 to 0.05 sec during limb CEU at 7 MHz in mice and 1.8 MHz in dogs. Destruction was also assessed by broadband signal generation during passive cavitation detection. Real-time CEU perfusion imaging with destruction-replenishment was then performed at 1.8 MHz in dogs using an MI of 0.1, 0.2, or 0.3. RESULTS: Sonazoid had a higher bulk modulus than Definity (66 ± 12 vs 29 ± 2 kPa, P = .02) and exhibited less inertial cavitation (destruction) at MIs ≥ 0.2. On in vivo CEU, maximal signal intensity increased incrementally with MI for both agents and was equivalent between agents except at an MI of 0.1 (60% and 85% lower for Sonazoid at 7 and 1.8 MHz, respectively, P < .05). However, on progressive shortening of the pulsing interval, Definity was nearly completely destroyed at MIs ≥ 0.2 at 1.8 and 7 MHz, whereas Sonazoid was destroyed only at 1.8 MHz at MIs ≥ 0.3. As a result, real-time CEU perfusion imaging demonstrated approximately fourfold greater enhancement for Sonazoid at an MI of 0.3 to 0.4. CONCLUSIONS: Robust signal enhancement during real-time CEU perfusion imaging of the limb is possible when using intermediate-power imaging coupled with a durable microbubble contrast agent.

On the combination of two visual cognition systems using combinatorial fusion.

When combining decisions made by two separate visual cognition systems, statistical means such as simple average (M 1) and weighted average (M 2 and M 3), incorporating the confidence level of each of these systems have been used. Although combination using these means can improve each of the individual systems, it is not known when and why this can happen. By extending a visual cognition system to become a scoring system based on each of the statistical means M 1, M 2, and M 3 respectively, the problem of combining visual cognition systems is transformed to the problem of combining multiple scoring systems. In this paper, we examine the combined results in terms of performance and diversity using combinatorial fusion, and study the issue of when and why a combined system can be better than individual systems. A data set from an experiment with twelve trials is analyzed. The findings demonstrated that combination of two visual cognition systems, based on weighted means M 2 or M 3, can improve each of the individual systems only when both of them have relatively good performance and they are diverse.

Identification and characterization of proteins involved in nuclear organization using Drosophila GFP protein trap lines.

BACKGROUND: Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31) gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp) is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl), a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology. CONCLUSIONS/SIGNIFICANCE: These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins.

A molecular dissection of the interaction between the transcription factor Gata-1 zinc finger and DNA.

The production of circulating blood cells from bone marrow stem cells during hematopoiesis is accompanied by overall changes in gene expression which cause production of required functional proteins, such as hemoglobin in erythroid cells, as well as control of cell growth, preventing apoptosis of differentiating cells. Hematopoietic gene regulation is controlled by several specific transcription factors, including the factor Gata-1, which is required for erythrocyte maturation. Based on contacts observed in the NMR structure of the cGata-1 binding domain in complex with DNA, the protein's key DNA interface is interesting in being quite hydrophobic in nature, due to the presence of three leucine side chains protruding toward the DNA. Given the T-rich composition of the GATA DNA binding site, it is possible that thymine's unique 5-methyl group may mediate some of these hydrophobic contacts to increase the stability of binding. The hypothesis that thymine methyl groups are important to the free energy of binding between Gata and DNA is tested by measuring binding of an oligonucleotide substrate in which individual thymine bases are substituted with uracil. To test for any important base-pair specific interactions which may be hydrogen-bonded in character, we have also assayed Gata binding to oligonucleotides with base analogs which cannot make hydrogen bonds. We report that out of the binding site's five thymine methyl groups, only one appeared to make a notable contribution to binding affinity, with removal causing a loss of less than 1kcal/mol of binding free energy. On the other hand, perturbing the potential hydrogen-bonding surface of the DNAs major groove was found to cause a larger decrease in binding affinity than removal of any of the thymine methyl groups, with a loss of 2-3kcal/mol of binding free energy.