Evaluation cardioprotective effects of atorvastatin in rats by real time myocardial contrast echocardiography.

BACKGROUND: The ability to assess myocardial perfusion in small animals is important, especially to investigate models of myocardial ischemia. Myocardial perfusion is usually assessed by postmortem techniques, eliminating the possibility of follow-up in intervention studies. The purpose of the study was to examine the feasibility of real time myocardial contrast echocardiography (MCE) to evaluate cardioprotective effects of atorvastatin in a rat model of acute ischemia-reperfusion injury. METHODS: The rats (n=15) underwent 20 minutes of mechanical left descending coronary artery (LAD) occlusion followed by 180 minutes of reperfusion. The animals received either atorvastatin (10 mg/kg), atorvastatin and the nitric oxide synthase (NOS)-inhibitor N-Nitro-L-Argininemethylester (L-NAME) (15 mg/kg), or vehicle. MCE was performed to assess the size of the perfusion defect and the myocardial signal intensities (A(max)) at the baseline, during occlusion, and during reperfusion. For comparison, the infarct size, risk area, and regional myocardial blood flow (MBF) were determined by the standard techniques as well. RESULTS: The dynamics of ischemia-reperfusion injury could be visualized serially by MCE. The infarct size-to-risk area ratio progressively increased during reperfusion and was markedly reduced in the atorvastatin group. Triphenyltetrazolium chloride (TTC) staining confirmed a 23% reduction in the infarct size by atorvastatin. The infarct size by MCE correlated well with the histological methods (r=0.86, P < 0.001). A(max) was reduced in the anterior segments during LAD occlusion (0.08 +/- 0.01 dB) compared to the baseline (2.9 +/- 0.4 dB), approached higher levels post revascularization of LAD (3.22 +/- 0.50 dB), but decreased during 180 minutes of reperfusion (2.32 +/- 0.40 dB). After 180 minutes of reperfusion, A(max) in the risk area was significantly higher in the atorvastain-treated group compared to the vehicle-treated group (2.32 +/- 0.40 dB vs 1.3 +/- 0.4 dB, P

Impact of microbubbles on shock wave-mediated DNA uptake in cells in vitro.

Gas-filled microbubbles have been successfully used as gene delivery reagents in combination with diagnostic ultrasound. Although shock wave exposure has been shown to transfect cells with naked DNA in vitro, it has not been tested whether the addition of microbubbles would augment DNA uptake under those conditions. Therefore, the aim of this study was to test the impact of microbubbles on transgene expression in vitro under shock wave exposure conditions. HEK 293 cells were treated with 60 or 120 pulses of shock waves at varying energy levels. Cells were mixed with either 100 microg/mL luciferase expressing plasmid DNA or with microbubbles that were produced with the same amount of this DNA. Cell death was evaluated after 1 h and transgene expression, after 24 h. In the presence of microbubbles, transgene expression was significantly higher (as much as 29-fold) relative to that obtained without microbubbles. Cells exposed to 120 pulses demonstrated higher transgene expression (as high as 2.7-fold) compared with cells exposed to 60 pulses. The use of microbubbles resulted in greater cell death, varying from 26% (low energy) to 78% (high energy). In conclusion, DNA-loaded microbubbles can significantly increase shock wave mediated gene transfer. However, this effect is associated with increased levels of cell destruction.

Cyclic variation of myocardial signal intensity in real-time myocardial perfusion imaging.

BACKGROUND: The presence of cyclic intensity variation during real-time myocardial perfusion imaging (RTPI) has been controversially discussed. We investigated whether cyclic intensity variation is systematically found during RTPI and whether such variations are related to regional functional parameters. METHODS: Intraoperative RTPI were obtained in 12 pigs before, during, and after left descending coronary artery occlusion with 60 mL/h SonoVue infusion. Furthermore, RTPI was performed in 14 patients after slow bolus injection of 0.7 mL of Optison. Instantaneous regional systolic to diastolic (S/D) myocardial intensity ratios were calculated after high mechanical index bubble destruction. S/D ratios were correlated with A- and beta-values, and fractional area shortening. RESULTS: Systematic cyclic S/D changes were present in both experimental settings showing significantly higher systolic values (animals, S/D 1.28 +/- 0.44; patients, S/D 1.25 +/- 0.7). Cyclic S/D variation was not related to fractional area shortening, or A- or beta-values (all r < 0.3, not significant). CONCLUSION: Consistent cyclic changes in myocardial contrast intensity can be measured both in intraoperative animals and in patients, showing higher systolic values. S/D ratios are not related to regional functional parameters.

Potential of gold-bound microtubules as a new ultrasound contrast agent.

Contrast agents based on gas-filled microspheres share the problem of time limited opacification due to low stability of microbubbles. The aim of this study was to test if gold-bound microtubules provide backscattering that allows microtubules to be potentially useful as an ultrasound (US) contrast agent. Gold colloids were immobilized on protein microtubule walls. Latex balloons were filled with gold-bound microtubules or conventional left heart contrast agent and were ultrasonographically imaged in fundamental and harmonic modes. Feasibility of anti-beta-tubulin antibody conjugation to gold-bound microtubules was confirmed using immune fluorescence analysis. Gold particles were successfully bound to microtubules. Contrast intensities in latex balloons filled with gold-bound microtubules (141 +/- 35) were comparable to those with Levovist (180 +/- 35) and did not decrease significantly during continuous US imaging for 20 min (135 +/- 34 vs. Levovist 5.0 +/- 2.0). Anti-beta-tubulin antibodies were successfully conjugated to gold-bound microtubules. Gold-bound microtubules provide a persistent contrast effect, suggesting their use as an ultrasonic contrast agent with the feasibility of antibody conjugation.

Myocardial left ventricular dysfunction in patients with systemic lupus erythematosus: new insights from tissue Doppler and strain imaging.

OBJECTIVE: Systemic lupus erythematosus (SLE) is associated with high cardiovascular morbidity and mortality. Cardiovascular involvement is frequently underestimated by routine imaging techniques. Our aim was to determine if new echocardiographic imaging modalities like tissue Doppler (TDI), strain rate (SRR), and strain (SRI) imaging detect abnormalities in left ventricular (LV) function in asymptomatic patients with SLE. METHODS: Sixty-seven young patients with SLE (mean age 42 +/- 10 yrs) without typical symptoms or signs of heart failure or angina, and a matched healthy control group (n = 40), underwent standard transthoracic echocardiography, TDI, SRR, and SRI imaging of the LV as well as assessment of disease characteristics. RESULTS: Despite findings within the normal range on routine standard 2-dimensional echocardiography, SLE was associated with significantly impaired systolic and diastolic myocardial velocities of the LV measured by TDI [mean global TDI: systolic (s): 2.9 +/- 0.9 vs 3.9 +/- 0.7 cm/s, p < 0.05; early (e): 4.3 +/- 1.5 vs 6.3 +/- 1.3 cm/s, p < 0.05; late (a): 2.9 +/- 0.8 vs 3.4 +/- 0.8 cm/s, p < 0.05; values +/- SD); SRR (s: -0.8 +/- 0.1 vs -1.1 +/- 0.1 s(-1); e: 1.1 +/- 0.2 vs 1.6 +/- 0.3 s(-1); a: 0.7 +/- 0.1 vs 1.0 +/- 0.2 s(-1); all p < 0.05); and SR (-15.11 +/- 2.2% vs -19.7 +/- 1.9%; p < 0.05) compared to the control group. Further, elevated disease activity, measured with the ECLAM and the SLEDAI score, resulted in significantly lower values for LV longitudinal function measured by SRR and SR, but not by TDI. CONCLUSION: SLE is associated with a significant impairment of systolic and diastolic LV longitudinal function in patients without cardiac symptoms. New imaging modalities provide earlier insight into cardiovascular involvement in SLE and seem to be superior to standard echocardiography to detect subclinical myocardial disease.

Ultrasound-targeted microbubble destruction augments protein delivery into testes.

OBJECTIVES: Gas-filled microbubbles have become an important tool as ultrasound contrast agents. In recent years, ultrasound-targeted microbubble destruction (UTMD) has evolved into a new tool for organ-specific gene and drug delivery. Although many studies have been performed in well-perfused target organs such as the heart or kidney, no study has yet investigated the feasibility of UTMD for delivery of bioactive substances in the testis. Thus, the aim of this study was to determine whether UTMD is a feasible and safe technique to deliver a reporter protein to the testes. METHODS: Different groups of rats received 2 microg of luciferase protein at varying protocols. One group received luciferase-loaded microbubbles infused intravenously while ultrasound was applied to the right testis. Another group received luciferase without microbubbles but with ultrasound applied to the right testis. Protein uptake was quantified by luciferase assay. Also, to rule out UTMD-induced damage, the testes were analyzed histologically. RESULTS: The testes that received ultrasound and luciferase-loaded microbubbles showed about twofold greater luciferase activity compared with testes without ultrasound or without microbubbles. No hemorrhage or microscopic damage was detected. CONCLUSIONS: The results of our study have shown that UTMD is a safe and feasible technique to augment delivery of bioactive substances to the testes.

Immediate and chronic effects of AV-delay optimization in patients with cardiac resynchronization therapy.

BACKGROUND: Acute changes of the AV-delay in CRT patients have a significant impact on hemodynamics. However, the chronic functional effects of AV-delay optimization have not been systematically examined despite of their potential role for chronic functional improvement. METHODS: Therefore, in this study we investigated whether optimization of AV-delay in CRT patients as assessed by echocardiographic measurement of the velocity time integral of the left ventricular outflow tract (LVOT-VTI) chronically changes (1) echocardiographic parameters of systolic and diastolic left ventricular function, (2) walking distance in the 6-min walk test, (3) levels of NT-proBNP and (4) quality of life as assessed by a standard questionnaire. 33 patients underwent optimization of AV-delay 31+/-8 weeks after initiation of CRT. Follow up (FU) was conducted 43+/-5 days later. RESULTS: E/Ea, the ratio of peak E-wave of mitral inflow and of TDI of the mitral annulus, significantly decreased immediately post-optimization (11+/-1 vs. 14+/-1 at baseline, p<0.05) and further decreased at FU (8+/-1, p<0.05 vs. immediately post-optimization) indicating improvement of diastolic function, while traditional parameters of diastolic function derived from pulse wave Doppler remained unchanged. There was a slight increase of LV-ejection fraction as assessed by echocardiography acutely after optimization (baseline: 25+/-2%, optimized: 28+/-1%, p<0.05), while LV-ejection fraction at FU did not differ from baseline. 6-min walk test improved from 449+/-17 m (baseline) to 475+/-17 m at FU (p<0.05). During this period NT-proBNP significantly decreased from 3193+/-765 ng/l to 2593+/-675 ng/l (p<0.05). Quality of life was unchanged at FU. CONCLUSION: This study demonstrates for the first time chronic functional improvement due to AV-delay optimization in patients with CRT.

Cardioprotective effects of the novel selective endothelin-A receptor antagonist BSF 461314 in ischemia-reperfusion injury.

OBJECTIVE: The aims of the study were to visualize the dynamics of ischemia-reperfusion injury by real-time myocardial contrast echocardiography and to investigate the cardioprotective effects of the novel endothelin-A receptor antagonist BSF 461314. BSF 461314 reduced infarct size by 47% and preserved microvascular integrity. Real-time myocardial contrast echocardiography allowed visualization of postischemic microvascular dysfunction and quantification of cardioprotective effects of selective endothelin antagonism. Blood flow index A x beta was reduced in anterior segments during ischemia compared with baseline (0.06 +/- 0.01 vs 0.98 +/- 0.2 dB/s) but was higher in the BSF 461314 group after 120 minutes of reperfusion (0.7 +/- 0.08 vs 0.3 +/- 0.05 dB/s, P = .015). Therefore, selective endothelin-A receptor antagonism improved microvascular integrity during postischemic reperfusion. Real-time myocardial contrast echocardiography accurately detected changes in microvascular reflow. BACKGROUND: Endothelin-1 is a potent vasoconstrictor and elevated in myocardial ischemia. The aims of the study were to examine cardioprotective effects of the novel selective endothelin-A receptor antagonist BSF 461314 and to visualize changes in the microvasculature by real-time myocardial contrast echocardiography (MCE). METHODS: A total of 16 open-chest pigs underwent 45 minutes of left anterior descending coronary artery occlusion followed by 120 minutes of reperfusion. A total of 1 mg/kg BSF 461314 or vehicle was given intravenously before reperfusion. Serial MCE was performed to assess changes in myocardial blood flow A x beta and perfusion defect size. Myocardial blood flow was measured by fluorescent microspheres and infarct size was measured by triphenyltetrazolium chloride tissue staining. RESULTS: Dynamics of infarct size expansion and tissue perfusion were correctly assessed by MCE. A x beta Was reduced in anterior segments during left anterior descending coronary artery occlusion (0.06 +/- 0.01 dB/s) compared with baseline (0.98 +/- 0.2 dB/s), approached higher levels postrecanalization (1.2 +/- 0.1 dB/s), but gradually decreased during reperfusion (0.3 +/- 0.05 dB/s, P < .01). After 120 minutes of reperfusion A (2.1 +/- 0.5 vs 1.0 +/- 0.6 dB, P < .03), beta (0.36 +/- 0.09/s vs 0.21 +/- 0.09/s, P = .01), and A x beta (0.7 +/- 0.08 vs 0.3 +/- 0.05 dB/s, P = .015) in the risk area were higher in the BSF 461314-treated group compared with vehicle indicating preserved myocardial perfusion. Triphenyltetrazolium chloride staining confirmed a 47% reduction in infarct size by BSF 461314. CONCLUSIONS: Selective endothelin-A receptor antagonism improved microvascular integrity during postischemic reperfusion. Real-time MCE allows visual and quantitative evaluation of dynamics of myocardial ischemia-reperfusion injury and monitoring of cardioprotective effects during pharmacologic interventions.

Detection of acute myocardial ischemia during pharmacological stress in graded coronary artery stenosis: analysis of left ventricular asynchrony using Fourier phase imaging in pigs.

BACKGROUND: Stress echocardiography is increasingly used to identify coronary artery disease, but quantitative techniques are required to improve the accuracy of this method. Current algorithms used to analyze wall motion usually neglect motion asynchrony that is found in acute ischemia. Fourier phase imaging of echocardiographic images may offer the possibility to detect asynchrony, but its feasibility with dobutamine stress echocardiography (DSE) is undefined. The aim of this study was to investigate whether the extent of left ventricular asynergy can be used to quantify the severity of regional myocardial dysfunction and to detect functionally significant coronary artery stenoses during DSE. METHODS: Regional wall motion abnormalities were induced by graded coronary stenoses (mild and severe) of the left anterior descending coronary artery (LAD) in seven open-chest anesthetized pigs. DSE (10-40 microg/kg/min) was performed under control conditions and during sustained ischemia. Coronary flow was measured under resting conditions and during maximal hyperemic response due to intravenous infusion of adenosine. Functional significance of stenoses was defined as mild when coronary flow reserve (CFR) was reduced but > 1.5 and severe when CFR was < or = 1.5. Echocardiographic cine loops were mathematically transformed using a first-harmonic Fourier algorithm displaying the sequence of wall motion as phase angles in parametric images and regional phase histograms. The phase difference (PD) of the first Fourier harmonic of posterior vs. anterior myocardial wall motion was calculated as an index of left ventricular asynchrony. Segmental fractional area shortening (FAS) and wall thickening (WT) as ratio of stress to rest served as a reference method of regional wall motion. RESULTS: The increase in FAS (1.62 +/- 0.6 vs. 0.42 +/- 0.2, p = 0.0002) and WT (1.92 +/- 0.5 to 0.3 +/- 1.1; p = 0.004) in anterior regions during DSE was significantly higher in the control group compared to severe ischemia but not compared to mild ischemia. During graded ischemia, profiles of phase angles were consistently modified, showing a delayed onset in regional contraction. The mean PD during DSE in the control group was 10.4 +/- 7 degrees. PD rose in mild ischemic segments (CFR > 1.5) to 28.9 +/- 10 degrees (p = 0.003) and to 89.6 +/- 25 degrees (p = 0.0002) in severely ischemic segments (CFR < or = 1.5). There was a significant inverse correlation between the PDs and WT (r = -0.87, p < 0.0001). Normal WT ratios yielded low phase angles while segmental phase angles increased with decreased WT. The intraobserver variability of phase analysis was 2.7 +/- 24 degrees (mean +/- 2SD). CONCLUSIONS: These results suggest that left ventricular asynchrony is an indicator of acute ischemia. Echocardiographic Fourier phase imaging is feasible to quantify wall motion displaying contraction sequence in a simple and objective format and is a promising approach for the clinical interpretation of stress echocardiograms.

Fourier phase analysis can be used to objectively analyze real-time myocardial contrast echocardiograms.

BACKGROUND: Real-time myocardial contrast echocardiography (MCE) is increasingly used to assess myocardial perfusion. However, objective methods for evaluating MCE are not yet widely available. We sought to validate the ability of Fourier analysis applied to MCE to assess serial changes in microvascular perfusion during coronary occlusion and reperfusion. METHODS: Six pigs underwent 45 min of left anterior descending coronary artery (LAD) occlusion followed by 120 min of reperfusion. Real time MCE was performed at baseline, during coronary occlusion, and at 5, 30, 60 and 120 min of reperfusion. Signal intensities from replenishment curves were fitted to an exponential function to obtain plateau SI (A) and the rate of SI rise (b). MCE images were mathematically transformed using a first-harmonic Fourier algorithm displaying the sequence of myocardial intensity changes as phase angles in parametric images. The phase angle difference (PD) of posterior vs. anterior region was calculated as an index of myocardial opacification heterogeneity and compared to MCE index of myocardial blood flow A x b. RESULTS: After initial hyperemia, a progressive reduction in flow was observed during reperfusion. During LAD occlusion signal intensities were significantly reduced in anterior regions (A x b = 0.02+/-0.01) compared to baseline (1.2+/-0.34, p < 0.01) defining risk areas and approached higher levels postrecanalization (A x b = 1.48+/-0.6) but gradually decreased during 120 min of reperfusion (A = 0.51+/-0.3, p < 0.01). Similarly, profiles of phase angles in LAD perfusion territorities were consistently modified during reperfusion. The mean PD at baseline was 18 degrees+/-15 degrees. PD decreased during coronary occlusion to -108 degrees+/-38 degrees, increased to 29 degrees+/-19 degrees postrecanalization but decreased to -61 degrees+/-35 degrees after 120 min of reperfusion. PD significantly correlated with A (r = 0.8, p < 0.0001) and b (r = 0.73, p < 0.0001). CONCLUSIONS: The progressive reduction in post-ischemic microvascular perfusion was accurately detected by real-time MCE. Fourier phase imaging is feasible to quantify dynamics of myocardial opacification in a simple and objective format and is a promising approach for the interpretation of contrast echocardiograms.

Fourier phase and amplitude analysis for automated objective evaluation of myocardial contrast echocardiograms.

AIMS: Objective methods for evaluating myocardial contrast echocardiography (MCE) are not yet widely available. We applied a Fourier analysis to myocardial contrast echocardiograms to identify myocardial perfusion defects. METHODS: Harmonic power-Doppler contrast echocardiograms were performed in 21 patients undergoing Tl-201-SPECT imaging and in 13 controls. Images were transformed using Fourier analysis to obtain phase of the first harmonic sinusoidal curve displayed as color coded sequence of myocardial intensity changes. Means and standard deviations of regional phase angles were measured. The method was validated in an in vitro model. A contrast filled latex balloon was imaged at different gain settings mimicking defined time-intensity curves. An intraoperative porcine infarction model served to prove feasibility of Fourier transformation to analyze real-time pulse inversion contrast echocardiography. RESULTS: In patients, phase imaging and intensity analysis showed focal areas with marked phase shifts (106 +/- 90 degrees) and heterogeneous distribution of phase angles (SD 66 +/- 17 degrees), correctly identifying 13/14 perfusion defects. The in vitro validation yielded increasing phase angles with increasing beta-values. This method was successfully applied to real-time MCE, identifying all infarction areas during occlusion of the left anterior descending artery. CONCLUSION: Phase analysis can be used to display dynamics of myocardial opacification.

Effects of different thrombolytic treatment regimen with abciximab and tirofiban on platelet aggregation and platelet-leukocyte interactions: a subgroup analysis from the GUSTO V and FASTER trials.

BACKGROUND: Due to considerably high rates of reocclusion under standard thrombolytic therapy GP IIb/IIIa inhibitors have been combined with thrombolytics to improve therapeutic outcomes. Potential reasons for arterial reocclusion may be increased platelet activation, interaction of platelets with other cell types such as leukocytes and inadequate drug dosing due to lack of ideal platelet monitoring. We compared combination therapy regimens consisting of GP IIb/IIIa inhibitors and thrombolytics with respect to platelet inhibition and platelet-leukocyte interactions. METHODS AND RESULTS: From the GUSTO V trial (standard rPA vs. reduced dose rPA and abciximab) and the FASTER trial (standard TNK-tPA vs. reduced dose TNK-tPA and tirofiban) 15 patients were monitored by platelet aggregometry, rapid platelet function assay (RPFA) and flow cytometry (FC). rPA alone (n = 5) caused initial increases in platelet aggregation. However, platelet aggregation was significantly (p < 0.05) and sufficiently (>80%) inhibited by abciximab/rPA (n = 5) and tirofiban/TNK-tPA (n = 5). The platelet inhibitory effect of tirofiban/TNK-tPA was more pronounced compared to abciximab/rPA with a significant difference after 2 h (p < 0.05). Tirofiban/TNK-tPA and abciximab/rPA caused decreases in platelet-leukocyte aggregates as well as in binding of specific antibodies to the platelet vitronectin receptor and P-selectin (p < 0.05, respect.). No differences among the treatment groups were seen with respect to antibody binding to MAC-1 and CD154/CD40 ligand. CONCLUSIONS: Taken together, GP IIb/IIIa inhibitors overcome the platelet activating effect of thrombolytics resulting in sufficient platelet inhibition. RPFA is a suitable monitoring tool to accurately assess platelet inhibition. Within the given combination treatment regimen tirofiban appears to be more effective compared to abciximab and to exert effects beyond the inhibition of GP IIb/IIIa.