Acoustic Activation Imaging With Intravenous Perfluoropropane Nanodroplets Results in Selective Bioactivation of the Risk Area.

BACKGROUND: Acoustically activatable perfluoropropane droplets (PD) can be formulated from commercially available microbubble preparations. Diagnostic transthoracic ultrasound frequencies have resulted in acoustic activation (AA) predominately within myocardial infarct zones (IZ). OBJECTIVE: We hypothesized that the AA area following acute coronary ischemia/reperfusion (I/R) would selectively enhance the developing scar zone, and target bioeffects specifically to this region. METHODS: We administered intravenous PD in 36 rats and 20 pigs at various stages of myocardial scar formation (30 minutes, 1 day, and 7 days post I/R) to determine what effect infarct age had on the AA within the IZ. This was correlated with histology, myeloperoxidase activity, and tissue nitrite activity. RESULTS: The degree of AA within the IZ in rats was not associated with collagen content, neutrophil infiltration, or infarct age. AA within 24 hours of I/R was associated with increased nitric oxide utilization selectively within the IZ (P < .05 compared with remote zone). The spatial extent of AA in pigs correlated with infarct size only when performed before sacrifice at 7 days (r = .74, P < .01). CONCLUSIONS: Acoustic activation of intravenous PD enhances the developing scar zone following I/R, and results in selective tissue nitric oxide utilization.

The START App: a web-based RNAseq analysis and visualization resource.

Summary: Transcriptional profiling using RNA sequencing (RNAseq) has emerged as a powerful methodology to quantify global gene expression patterns in various contexts from single cells to whole tissues. The tremendous amount of data generated by this profiling technology presents a daunting challenge in terms of effectively visualizing and interpreting results. Convenient and intuitive data interfaces are critical for researchers to easily upload, analyze and visualize their RNAseq data. We designed the START (Shiny Transcriptome Analysis Resource Tool) App with these requirements in mind. This application has the power and flexibility to be resident on a local computer or serve as a web-based environment, enabling easy sharing of data between researchers and collaborators. Availability and Implementation: Source Code for the START App is written entirely in R and can be freely available to download at https://github.com/jminnier/STARTapp with the code licensed under GPLv3. It can be launched on any system that has R installed. The START App is also hosted on https://kcvi.shinyapps.io/START for researchers to temporarily upload their data. Contact: minnier@ohsu.edu

Effect of Ambient Conditions on Acoustic Activation of the Perfluoropropane Droplets Within the Infarct Zone.

BACKGROUND: Acoustically activated perfluoropropane droplets (PD) formulated from lipid encapsulated microbubble preparations produce a delayed myocardial contrast enhancement that preferentially highlights the infarct zones (IZ). Since activation of PDs may be temperature sensitive, it is unclear what effect body temperature (BT) has on acoustic activation (AA). OBJECTIVE: We sought to determine whether the microvascular retention and degree of myocardial contrast intensity (MCI) would be affected by BT at the time of intravenous injection. METHODS: We administered intravenous (IV) PD in nine rats following 60 min of ischemia followed by reperfusion. Injections in these rats were given at temperatures above and below 36.5°C, with high MI activation in both groups at 3 or 6 min following IV injection (IVI). In six additional rats (three in each group), IV PDs were given only at one temperature (<36.5°C or ≥36.5°C), permitting a total of 12 comparisons of different BT. Differences in background subtracted MCI at 3-6 min post-injection were compared in the infarct zone (IZ) and remote zone (RZ). Post-mortem lung hematoxylin and eosin (H&E) staining was performed to assess the effect potential thermal activation on lung tissue. RESULTS: Selective MCI within the IZ was observed in 8 of 12 rats who received IVI of PDs at <36.5°C, but none of the 12 rats who had IVI at the higher temperature (p < 0.0001). Absolute MCI following droplet activation was significantly higher in both the IZ and RZ when given at the lower BT. H&E indicated significant red blood extravasation in 5/7 rats who had had IV injections at higher BT, and 0/7 rats who had IV PDs at <36.5°C. CONCLUSIONS: Selective IZ enhancement with AA of intravenous PDs is possible, but temperature sensitive. Thermal activation appears to occur when PDs are given at higher temperatures, preventing AA, and increasing unwanted bioeffects.

Delayed Echo Enhancement Imaging to Quantify Myocardial Infarct Size.

BACKGROUND: Perfluoropropane droplets formulated from commercial microbubbles exhibit different acoustic characteristics than their parent microbubbles, most likely from enhanced endothelial permeability. This enhanced permeability may permit delayed echo-enhancement imaging (DEEI) similar to delayed enhancement magnetic resonance imaging (DE-MRI). We hypothesized this would allow detection and quantification of myocardial scar. METHODS: In 15 pigs undergoing 90 minutes of left anterior descending ischemia by either balloon (n = 13) or thrombotic occlusion (n = 2), DE-MRI was performed at 2-24 days postocclusion. Delayed echo-enhancement imaging was performed at 2-4 minutes following an intravenous injection of 1 mL of 50% Definity (Lantheus Medical) compressed into 180 nm droplets; DEEI was attempted in all pigs with single-pulse harmonic imaging at 1.7 transmit/3.4 MHz receive. Myocardial defects observed with DEEI were quantified (percentage of infarct area) and compared with DE-MRI as well as postmortem staining. In six pigs, multipulse low-mechanical index (MI) fundamental nonlinear imaging (FNLI) with intermittent high-MI impulses was performed to determine whether droplet activation within the infarct zone was achievable with a longer pulse duration. RESULTS: The range of infarct size area by DE-MRI ranged from 0% to 46% of total left ventricular area. Single-pulse harmonic imaging detected a contrast defect that correlated closely with infarct area by DE-MRI (r = 0.81, P = .0001). The FNLI high-MI impulses resulted in droplet activation in both the infarct and normal zones. Harmonic subtraction of the FNLI images resulted in infarct zone enhancement that also correlated closely with infarct size (r = 0.83; P = .04). Droplets were observed on postmortem transmission electron microscopy within myocytes of the infarct and remote normal zone. CONCLUSION: Intravenously Definity nanodroplets can be utilized to detect and quantify infarct zone at the bedside using DEEI techniques.

Screening entire healthcare system ECG database: Association of deep terminal negativity of P wave in lead V1 and ECG referral with mortality.

BACKGROUND: Each encounter of asymptomatic individuals with the healthcare system presents an opportunity for improvement of cardiovascular disease (CVD) awareness and sudden cardiac death (SCD) risk assessment. ECG sign deep terminal negativity of the P wave in V1 (DTNPV1) was shown to be associated with an increased risk of SCD in the general population. OBJECTIVE: To evaluate association of DTNPV1 with all-cause mortality and newly diagnosed atrial fibrillation (AFib) in the large tertiary healthcare system patient population. METHODS: Retrospective double cohort study compared two levels of exposure (automatically measured amplitude of P-prime (Pp) in V1): DTNPV1 (Pp from -100µV to -200µV) and ZeroPpV1 (Pp=0). An entire healthcare system (2010-2014) ECG database was screened. Medical records of children and patients with previously diagnosed AFib/atrial flutter (AFl), implanted pacemaker or cardioverter-defibrillator were excluded. DTNPV1 (n=3,413) and ZeroPpV1 (n=3,405) cohorts were matched by age and sex. Primary outcome was all-cause mortality. Secondary outcomes were newly diagnosed AFib/AFl. Median follow-up was 2.5 y. RESULTS: DTNPV1 was associated with all-cause mortality (HR 1.95(1.64-2.31); P<0.0001) and newly diagnosed AFib (HR 1.29(1.04-1.59); P=0.021) after adjustment for CVD, comorbidities, other ECG parameters, medications, and index ECG referral. Index ECG referral by a cardiologist was independently associated with 34% relative risk reduction of mortality (HR 0.66(0.52-0.84); P=0.001), as compared to ECG referral by a non-cardiologist. CONCLUSION: DTNPV1 is independently associated with twice higher risk of all-cause death, as compared to patients without P prime in V1. Life-saving effect of the index ECG referral by a cardiologist requires further study.

Role of collateral blood flow in the apparent disparity between the extent of abnormal wall thickening and perfusion defect size during acute myocardial infarction and demand ischemia.

OBJECTIVES: The aim of this study was to test the hypothesis that the apparent disparity between the circumferential extent of abnormal wall thickening (WT) and that of infarct size (IS) at rest or size of ischemic zone (IZ) during demand ischemia (DI) is principally due to the effects of collateral blood flow (CollBF). BACKGROUND: A disparity has been reported between the circumferential extent of abnormal WT and that of IS at rest or IZ size during DI. METHODS: Wall thickening and CollBF were measured in 18 dogs: at 6 h after coronary occlusion (Group 1, n = 6), and during 40 microg x kg x min(-1) of dobutamine in the presence of either one-vessel (Group 2, n = 6) or two-vessel stenosis (Group 3, n = 6). RESULTS: The apparent overestimation of the IS by the circumferential extent of abnormal WT was due to intermediate levels of CollBF in border zones within the risk area that had escaped necrosis. Although reduced, WT in these regions was commensurate with the level of flow. Similarly, during DI, regions within the IZ exhibiting the worst WT in Group 2 and 3 dogs were those not supplied by CollBF. The regions supplied by CollBF had intermediate WT, which was also commensurate with the level of flow. Only in two Group 3 dogs was tethering seen in small, normally perfused regions that were interspersed between two large IZ. Excluding these few tethered regions, data from different myocardial regions (infarcted, ischemic, CollBF dependent, and normal) were described by a single relation: y = 57(1 - e([-0.72(x - 0.06)])) (r = 0.80, p < 0.001). CONCLUSIONS: Myocardial regions at the margins of ischemic territories contribute to the apparent disparity between the circumferential extent of abnormal WT and IS or IZ during DI. In most circumstances, these regions are supplied by collaterals and their WT is commensurate with the degree of myocardial blood flow. The apparent disparity between the circumferential extent of WT and ischemia is rarely due to myocardial tethering, which is seen only in some instances of multi-vessel disease where a small normal region is interspersed between two large IZs.

Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to alpha(v)beta3.

BACKGROUND: Angiogenesis is a critical determinant of tumor growth and metastasis. We hypothesized that contrast-enhanced ultrasound (CEU) with microbubbles targeted to alpha(v)-integrins expressed on the neovascular endothelium could be used to image angiogenesis. METHODS AND RESULTS: Malignant gliomas were produced in 14 athymic rats by intracerebral implantation of U87MG human glioma cells. On day 14 or day 28 after implantation, CEU was performed with microbubbles targeted to alpha(v)beta3 by surface conjugation of echistatin. CEU perfusion imaging with nontargeted microbubbles was used to derive tumor microvascular blood volume and blood velocity. Vascular alpha(v)-integrin expression was assessed by immunohistochemistry, and microbubble adhesion was characterized by confocal microscopy. Mean tumor size increased markedly from 14 to 28 days (2+/-1 versus 35+/-14 mm2, P<0.001). Tumor blood volume increased by approximately 35% from day 14 to day 28, whereas microvascular blood velocity decreased, especially at the central portions of the tumors. On confocal microscopy, alpha(v)beta3-targeted but not control microbubbles were retained preferentially within the tumor microcirculation. CEU signal from alpha(v)beta3-targeted microbubbles in tumors increased significantly from 14 to 28 days (1.7+/-0.4 versus 3.3+/-1.0 relative units, P<0.05). CEU signal from alpha(v)beta3-targeted microbubbles was greatest at the periphery of tumors, where alpha(v)-integrin expression was most prominent, and correlated well with tumor microvascular blood volume (r=0.86). CONCLUSIONS: CEU with microbubbles targeted to alpha(v)beta3 can noninvasively detect early tumor angiogenesis. This technique, when coupled with changes in blood volume and velocity, may provide insights into the biology of tumor angiogenesis and be used for diagnostic applications.

Feasibility of Using Electronic Medical Record Data for Tracking Quality Indicators in Adults with Congenital Heart Disease.

BACKGROUND: In order to determine the feasibility of tracking quality of care in adults with congenital heart disease (ACHD), we aimed to estimate the availability of relevant data in electronic medical records (EMR) used in North American ACHD centers. METHODS: Previously proposed quality indicators (QIs) were reviewed to consider what types of data would be required for each. ACHD program directors were surveyed about the nature of electronic data in existing EMRs. From the survey, the availability of data types needed for the denominator and numerator of each QI were estimated, and an overall estimate of data availability was calculated for each QI. These estimates were adjusted by the sensitivity of identifying the patients through administrative codes. Analysis was repeated for scenarios in which various data type estimates were hypothetically dropped by half to determine the overall impact of each data type. RESULTS: A total of 64 ACHD program directors responded to the survey. Of 55 QIs, average estimated data availability was 67%. QIs for tetralogy of Fallot had the highest estimated data availability (mean 88%), whereas those for atrial septal defect were lowest (mean 23%), reflecting both the need for interpretation of imaging studies and the lower reliability of billing codes for identification of ACHD patients. QIs with highest estimates were based largely on administrative data, which had the biggest impact on overall estimates. QIs needing interpretation of imaging findings had the lowest estimates, as well as certain overuse measures. CONCLUSIONS: For a wide range of ACHD programs, data for proposed QIs based on administrative data are most likely to be obtainable through EMR. Data related to imaging interpretation or overuse measures are least likely. Our findings can inform future efforts to establish registry efforts or data reporting tools to track these indicators.

Molecular imaging identifies regions with microthromboemboli during primary angioplasty in acute coronary thrombosis.

UNLABELLED: Microthromboemboli (MTE) may contribute to the no-reflow phenomenon in acute myocardial infarction (AMI) either spontaneously or after primary percutaneous transluminal coronary angioplasty (PTCA). We hypothesized that myocardial MTE in acute coronary syndromes can be identified on imaging by in vivo (99m)Tc labeling of the coronary thrombus with a compound that binds to the glycoprotein IIb/IIIa present on activated platelets (DMP-444). METHODS: Fifteen dogs underwent left anterior descending coronary artery (LAD) injury in to produce thrombus, whereas 5 control dogs had LAD ligation. Before recanalization, the risk area (RA) and myocardial blood flow (MBF) were measured, and in vivo thrombus labeling was performed using (99m)Tc-labeled DMP-444. Nine of the 15 LAD injury dogs had occlusive thrombus on angiography and underwent PTCA. MBF measurements were repeated 30 and 60 min after recanalization, and (99m)Tc autoradiography (hot spot imaging) was performed ex vivo to determine the extent and magnitude of MTE. RESULTS: The ratio of hot spot size to RA size was higher in the 9 LAD injury dogs with thrombus compared with the 6 dogs with no thrombus (90% +/- 22% vs. 42% +/- 16%; P = 0.005). In control dogs, this ratio was significantly lower (29% +/- 11%; P = 0.05). (99m)Tc activity within the RA was higher in 8 of the 15 coronary injury dogs with AMI compared with those without AMI (1.8 +/- 0.48 vs. 1.24 +/- 0.22; P = 0.02). CONCLUSION: MTE can be detected and quantified after primary PTCA. The infarct size is proportional to the magnitude and extent of MTE, indicating that MTE may contribute to the AMI. Thus, in vivo thrombus labeling during reperfusion may provide important information in patients with AMI that may lead to better adjuvant therapy during PTCA.

Microvascular rheology of Definity microbubbles after intra-arterial and intravenous administration.

The microvascular rheology and extent of pulmonary retention of second-generation microbubble ultrasound contrast agents has not previously been well characterized. We assessed the microvascular behavior of Definity, a lipid-shelled microbubble agent containing perfluoropropane gas, using intravital microscopy of either rat spinotrapezius muscle or mouse cremaster muscle. Immediately after intra-arterial injection, which was performed to model pulmonary retention, larger microbubbles (> 5 microm) were entrapped within small arterioles and capillaries. The retention fraction of microbubbles was low (1.2% +/- 0.1%) and entrapment was transient (85% dislodged by 10 minutes), resulting in no adverse hemodynamic effects. Leukocyte or platelet adhesion at the site of entrapment was not seen. After intravenous injection, no microbubble entrapment was observed and the velocities of microbubbles in arterioles, venules, and capillaries correlated well with those of red blood cells. We conclude that after intravenous injection and pulmonary passage, the microvascular rheology of Definity microbubbles is similar to that of red blood cells. Microbubble entrapment within the pulmonary microcirculation after venous injection should be negligible and transient. These findings are important for establishing the safety of this agent.

Automated, nonrigid alignment of clinical myocardial contrast echocardiography image sequences: comparison with manual alignment.

Analysis of myocardial contrast echocardiography (MCE) images is currently done by manual techniques. The development of computationally efficient methods for aligning images provides an important first step toward the automation of MCE analysis. This is challenging because a nonrigid transformation correction is required. In this paper, we evaluate a state-of-the-art nonrigid alignment method on clinical MCE image sequences (n = 58) acquired on patients during rest and dipyridamole stress, using both B-mode intermittent ultraharmonic (IUH) imaging and real-time myocardial perfusion imaging (RTMPI). Using manual alignment as the reference, we show quantitatively that the automated method aligns images as well as a human observer. However, the new method is faster and more reliable than manual alignment and removes the need for an experienced physician to perform it. The automated technique can be used for quick poststudy off-line analysis and has the potential to be incorporated into an ultrasound machine.

Cyclic variation in ultrasonic myocardial integrated backscatter is due to phasic changes in the number of patent myocardial microvessels.

OBJECTIVE: We tested the hypothesis that the cyclic variation in ultrasonic myocardial integrated backscatter (IBS) is due to cardiac contraction-induced changes in the number of patent myocardial microvessels. METHODS: We performed experiments in open-chest dogs in which we increased the number of patent myocardial microvessels without changing cardiac contraction. We achieved this either by direct intracoronary administration of adenosine (group 1; n = 10) or by producing a noncritical coronary stenosis (group 2; n = 7). RESULTS: At baseline, IBS was lowest in systole and highest in diastole. This cyclic variation in IBS was closely associated with the phasic changes in myocardial blood volume that were measured with myocardial contrast echocardiography. During adenosine administration, the diastolic IBS increased from -18.8 +/- 6.5 to -17.5 +/- 6.1 dB (P = .002), with an associated increase in the difference between the systolic and diastolic IBS from 3.8 +/- 1.1 to 4.6 +/- 1.0 dB (P = .009). After a noncritical stenosis was produced, diastolic IBS also increased from -26.6 +/- 8.3 to -25.2 +/- 7.3 dB (P = .001), with an associated increase in the difference between the systolic and diastolic IBS from 3.7 +/- 1.2 to 5.0 +/- 1.0 dB (P = .02). No change in IBS was noted in the bed that did not receive adenosine or the bed that had a stenosis. CONCLUSIONS: The variation in IBS during the cardiac cycle is closely associated with the phasic changes in myocardial blood volume seen during cardiac contraction. When the number of patent myocardial arterioles is increased via adenosine or placement of a noncritical stenosis, diastolic IBS increases with a concomitant increase in IBS cyclic variation. These results may have important clinical applications for the noninvasive diagnosis of noncritical coronary stenosis at rest.

Automated quantification of the spatial extent of perfusion defects and viability on myocardial contrast echocardiography.

The spatial extent of hypoperfusion or viability is important in the treatment of patients with coronary artery disease. We hypothesized that computerized pixel intensity threshold analysis (PITA) could be used for the automated analysis of perfusion defect size during myocardial contrast echocardiography (MCE). For calibration studies, MCE was performed in 6 dogs undergoing ischemia and reperfusion. Infarct size was determined by PITA, which automatically calculates the percentage of pixels within the myocardium that fail to exceed a predetermined threshold of maximum contrast enhancement. A threshold of 10% of maximum yielded infarct sizes that most closely correlated with those determined by histologic staining. For clinical validation, MCE was performed in 30 patients with acute myocardial infarction before primary percutaneous coronary intervention (PCI) for measurement of risk area; and within 5 days and at 4 weeks after PCI to determine infarct size. The defect size by PITA with a 10% threshold value closely correlated with those measured by expert reader planimetry on background-subtracted color-coded image sets (r = 0.95, P < .001). We conclude that automated analysis of perfusion defect size on MCE is possible by PITA. This technique may be useful for rapid and objective analysis of the extent of ischemia and viability, and for clinical experimentation where accurate and sequential analysis of perfusion defect size is imperative.