Spatial distributions of pericellular stiffness in natural extracellular matrices are dependent on cell-mediated proteolysis and contractility.

Bulk tissue stiffness has been correlated with regulation of cellular processes and conversely cells have been shown to remodel their pericellular tissue according to a complex feedback mechanism critical to development, homeostasis, and disease. However, bulk rheological methods mask the dynamics within a heterogeneous fibrous extracellular matrix (ECM) in the region proximal to a cell (pericellular region). Here, we use optical tweezers active microrheology (AMR) to probe the distribution of the complex material response function (α=α'+α″, in units of µm/nN) within a type I collagen ECM, a biomaterial commonly used in tissue engineering. We discovered cells both elastically and plastically deformed the pericellular material. α' is wildly heterogeneous, with 1/α' values spanning three orders of magnitude around a single cell. This was observed in gels having a cell-free 1/α' of approximately 0.5nN/µm. We also found that inhibition of cell contractility instantaneously softens the pericellular space and reduces stiffness heterogeneity, suggesting the system was strain hardened and not only plastically remodeled. The remaining regions of high stiffness suggest cellular remodeling of the surrounding matrix. To test this hypothesis, cells were incubated within the type I collagen gel for 24-h in a media containing a broad-spectrum matrix metalloproteinase (MMP) inhibitor. While pericellular material maintained stiffness asymmetry, stiffness magnitudes were reduced. Dual inhibition demonstrates that the combination of MMP activity and contractility is necessary to establish the pericellular stiffness landscape. This heterogeneity in stiffness suggests the distribution of pericellular stiffness, and not bulk stiffness alone, must be considered in the study of cell-ECM interactions and design of complex biomaterial scaffolds. STATEMENT OF SIGNIFICANCE: Collagen is a fibrous extracellular matrix (ECM) protein widely used to study cell-ECM interactions. Stiffness of ECM has been shown to instruct cells, which can in turn modify their ECM, as has been shown in the study of cancer and regenerative medicine. Here we measure the stiffness of the collagen microenvironment surrounding cells and quantitatively measure the dependence of pericellular stiffness on MMP activity and cytoskeletal contractility. Competent cell-mediated stiffening results in a wildly heterogeneous micromechanical topography, with values spanning orders of magnitude around a single cell. We speculate studies must consider this notable heterogeneity generated by cells when testing theories regarding the role of ECM mechanics in health and disease.

Characterization of hydrogel microstructure using laser tweezers particle tracking and confocal reflection imaging.

Hydrogels are commonly used as extracellular matrix mimetics for applications in tissue engineering and increasingly as cell culture platforms with which to study the influence of biophysical and biochemical cues on cell function in 3D. In recent years, a significant number of studies have focused on linking substrate mechanical properties to cell function using standard methodologies to characterize the bulk mechanical properties of the hydrogel substrates. However, current understanding of the correlations between the microstructural mechanical properties of hydrogels and cell function in 3D is poor, in part because of a lack of appropriate techniques. Here we have utilized a laser tracking system, based on passive optical microrheology instrumentation, to characterize the microstructure of viscoelastic fibrin clots. Trajectories and mean square displacements were observed as bioinert PEGylated (PEG: polyethylene glycol) microspheres (1, 2 or 4.7 µm in diameter) diffused within confined pores created by the protein phase of fibrin hydrogels. Complementary confocal reflection imaging revealed microstructures comprised of a highly heterogeneous fibrin network with a wide range of pore sizes. As the protein concentration of fibrin gels was increased, our quantitative laser tracking measurements showed a corresponding decrease in particle mean square displacements with greater resolution and sensitivity than conventional imaging techniques. This platform-independent method will enable a more complete understanding of how changes in substrate mechanical properties simultaneously influence other microenvironmental parameters in 3D cultures.

Controlled ablation of microtubules using a picosecond laser.

The use of focused high-intensity light sources for ablative perturbation has been an important technique for cell biological and developmental studies. In targeting subcellular structures many studies have to deal with the inability to target, with certainty, an organelle or large macromolecular complex. Here we demonstrate the ability to selectively target microtubule-based structures with a laser microbeam through the use of enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) variants of green fluorescent protein fusions of tubule. Potorous tridactylus (PTK2) cell lines were generated that stably express EYFP and ECFP tagged to the alpha-subunit of tubulin. Using microtubule fluorescence as a guide, cells were irradiated with picosecond laser pulses at discrete microtubule sites in the cytoplasm and the mitotic spindle. Correlative thin-section transmission electron micrographs of cells fixed one second after irradiation demonstrated that the nature of the ultrastructural damage appeared to be different between the EYFP and the ECFP constructs suggesting different photon interaction mechanisms. We conclude that focal disruption of single cytoplasmic and spindle microtubules can be precisely controlled by combining laser microbeam irradiation with different fluorescent fusion constructs. The possible photon interaction mechanisms are discussed in detail.

Impact of a home-based walking and resistance training program on quality of life in patients with heart failure.

Patients with heart failure (HF) often have profound activity limitations and diminished quality of life (QOL) due to symptoms of dyspnea and fatigue. Although recent studies demonstrate positive physiologic and psychological benefits of low to moderate intensity, supervised, aerobic exercise training performed 3 to 5 days/ week for 20 to 40 minutes' duration, in a monitored setting, the efficacy of a home-based exercise program combining endurance and resistance exercise on symptoms and QOL, are unknown. This randomized controlled study examined the efficacy, safety, and adherence rates of a 3-month home-based combined walking and resistance exercise program on symptoms and QOL in 40 women and men aged 30 to 76 years with New York Heart Association class II to III HF. Baseline and 3-month evaluations consisted of a chronic HF questionnaire to assess symptoms and QOL and exercise capacity by symptom-limited treadmill exercise test with respiratory gas analysis. The exercise intervention improved fatigue (p = 0.02), emotional function (p = 0.01), and mastery (p = 0.04). Overall exercise adherence was excellent (90%) and there were no reported adverse events. A moderate intensity home-based combined walking and resistance program for patients with class II to III HF is safe and effective in reducing symptoms and improving QOL.

Effects of the repeated administration of adenosine and heparin on myocardial perfusion in patients with chronic stable angina pectoris.

The mechanism by which ischemia stimulates angiogenesis is unknown. Adenosine is released during myocardial ischemia and may be a mediator of this process. Experimental data suggest that heparin may enhance this effect. The purpose of this open-labeled, placebo-controlled trial was to determine whether repeated intravenous administration of adenosine and heparin could mimic physiologic angiogenesis and reduce the amount of exercise-induced myocardial ischemia in patients with coronary artery disease. Subjects with chronic stable angina refractory to conventional medical therapy and not suitable for revascularization received either adenosine (140 microg/kg/min for 6 minutes) and heparin (10,000 U bolus), (n = 14), or placebo, (n = 7) daily for 10 days. All patients underwent baseline and follow-up exercise testing with thallium-201 single-photon emission computed tomography myocardial perfusion imaging. A semiquantitative assessment of the extent and severity of the perfusion abnormalities was calculated by 2 blinded investigators. There was no significant change in exercise duration or in the peak heart rate systolic blood pressure product associated with adenosine and heparin compared with placebo treatment. There was, however, a 9% reduction in the extent (60.6 +/- 4.0 vs 54.9 +/- 4.1, p = 0.03) and a 14% improvement in severity (41.5 +/- 3.2 vs 35.7 +/- 2.9, p = 0.01) of the myocardial perfusion abnormalities seen in patients who received adenosine and heparin compared with placebo. Thus, in this pilot study, repeated administration of adenosine and heparin reduced the amount of exercise-induced ischemia in patients with chronic stable angina refractory to conventional treatment.

Intrinsic dual-energy processing of myocardial perfusion images.

UNLABELLED: We have developed a software-based method for processing dual-energy 201TI SPECT emission projection data with the goal of calculating a spatially dependent index of the local impact of gamma-ray attenuation. We refer to this method as intrinsic dual-energy processing (IDEP). METHODS: IDEP exploits the differential attenuation of lower energy emissions (69-83 keV) and higher energy emissions (167 keV) resulting from the decay of 201TI to characterize the relative degree of low-energy gamma-ray attenuation throughout the myocardium. In particular, IDEP can be used to estimate the relative probability that a low-energy gamma-ray emitted from a particular region of the myocardium is detected during the acquisition of SPECT projection data. Studies on phantoms and healthy human volunteers were performed to determine whether the IDEP method yielded detection probability images with systematic structure visible above the noise of these images and whether the systematic structure in the detection probability images could be rationalized physically. In patient studies, the relative regional detection probabilities were applied qualitatively to determine the likely effects of attenuation on the distribution of mapped photon emissions. RESULTS: Measurements of the detection probability in uniform phantoms showed excellent agreement with those obtained from computer simulations for both 180 degrees and 360 degrees acquisitions. Additional simulations with digital phantoms showed good correlation between IDEP-estimated detection probabilities and calculated detection probabilities. In patient studies, the IDEP-derived detection probability maps showed qualitative agreement with known nonuniform attenuation characteristics of the human thorax. When IDEP data were integrated with the findings on the emission scan, the correlation with coronary anatomy (known in 6 patients and hypothesized on the basis of clinical and electrocardiographic parameters in 5 patients) was improved compared with evaluating the mapped emission image alone. CONCLUSION: The IDEP method has the potential to characterize the attenuation properties of an object without use of a separate transmission scan. Coupled with the emission data, it may aid coronary diagnosis.

Ventricular contraction abnormalities in dilated cardiomyopathy: effect of biventricular pacing to correct interventricular dyssynchrony.

OBJECTIVE: To measure ventricular contractile synchrony in patients with dilated cardiomyopathy (DCM) and to evaluate the effects of biventricular pacing on contractile synchrony and ejection fraction. BACKGROUND: Dilated cardiomyopathy is characterized by abnormal ventricular activation and contraction. Biventricular pacing may promote a more coordinated ventricular contraction pattern in these patients. We hypothesized that biventricular pacing would improve synchrony of right ventricular and left ventricular (RV/LV) contraction, resulting in improved ventricular ejection fraction. METHODS: Thirteen patients with DCM and intraventricular conduction delay underwent multiple gated equilibrium blood pool scintigraphy. Phase image analysis was applied to the scintigraphic data and mean phase angles computed for the RV and LV. Phase measures of interventricular (RV/LV) synchrony were computed in sinus rhythm and during atrial sensed biventricular pacing (BiV). RESULTS: The degree of interventricular dyssynchrony present in normal sinus rhythm correlated with LV ejection fraction (r = -0.69, p < 0.01). During BiV, interventricular contractile synchrony improved overall from 27.5 +/- 23.1 degrees to 14.1 +/- 13 degrees (p = 0.01). The degree of interventricular dyssynchrony present in sinus rhythm correlated with the magnitude of improvement in synchrony during BiV (r = 0.83, p < 0.001). Left ventricular ejection fraction increased in all thirteen patients during BiV, from 17.2 +/- 7.9% to 22.5 +/- 8.3% (p < 0.0001) and correlated significantly with improvement in RV/LV synchrony during BiV (r = 0.86, p < 0.001). CONCLUSIONS: Dilated cardiomyopathy with intraventricular conduction delay is associated with significant interventricular dyssynchrony. Improvements in interventricular synchrony during biventricular pacing correlate with acute improvements in LV ejection fraction.

Long-term estrogen replacement therapy is associated with improved exercise capacity in postmenopausal women without known coronary artery disease.

BACKGROUND: Short-term estrogen administration improves vasodilation and has been shown to improve exercise capacity. However, it is unknown whether long-term estrogen replacement therapy is associated with improved exercise capacity in postmenopausal women without known coronary artery disease. METHODS AND RESULTS: We studied 248 postmenopausal women without known coronary artery disease (mean age 63.5 years); 158 (64%) were current or past hormone replacement therapy (HRT) users and 108 (44%) were current users of HRT. Attributes potentially affecting exercise capacity and cardiac risk factors were carefully measured. These included duration of estrogen replacement therapy, all variables in the Framingham risk index, physical activity level, body mass index, waist-to-hip ratio, presence of osteoporosis, and family history of heart disease. We measured maximal oxygen uptake (MVO (2)) and anaerobic threshold as objective markers of exercise capacity. The relation between exercise capacity and use of HRT was analyzed with the use of logistic regression, controlling for confounding variables. We found that fitness, as measured by MVO (2) and anaerobic threshold, was significantly greater in women who had used HRT currently or in the past compared with women who had never used HRT. This difference in fitness was not confounded by age or physical activity level. CONCLUSIONS: Estrogen replacement therapy is associated with increased exercise capacity as measured by MVO (2) and anaerobic threshold in postmenopausal women without coronary artery disease. This finding is consistent with the beneficial effect of short-term estrogen administration on improved endothelium-dependent and endothelium-independent vasodilation.

Potential added value of three-dimensional reconstruction and display of single photon emission computed tomographic gated blood pool images.

BACKGROUND: Single photon emission computed tomographic (SPECT) acquisition provides potential advantages for blood pool imaging. However, the method has been little applied. METHODS: An improved method of three-dimensional (3-D) reconstruction and display of SPECT equilibrium blood pool scintigrams and related phase data was developed. Dynamic slices and volume-rendered dynamic 3-D images were displayed. Images were viewed from each of 34 solid angles referenced to a sphere surrounding the reconstruction field. Each image pixel was "painted" with intensity-coded regional amplitude and color-coded for its phase angle. The method was applied to evaluate the cardiac anatomy, regional contraction, and related conduction sequence at rest in 17 patients. Twelve had normal left ventricular function including 7 patients with minimal septal preexcitation. Five patients had abnormal left ventricular function, including 2 with left bundle branch block. RESULTS: Slices contained all of the functional information, but necessary data integration was time-consuming and evaluation of chamber size and anatomy was difficult. Three-dimensional projection images condensed and integrated the data, presenting new vantage points on anatomy, contraction, and conduction not otherwise available in the clinically limited angulations of planar images. This provided excellent visual separation of cardiac chambers with full and increased visualization of right and left ventricular wall motion in all segments compared with the conventional projections acquired clinically (p < 0.05). Atria and great vessels were well separated with evident size and function. Phase-angle progression paralleled the electrocardiogram, permitting bypass pathway localization and the direct noninvasive localization of posteroseptal pathways. CONCLUSIONS: The 3-D method permits greater access to and utilization of SPECT blood pool image data. It suggests specific advantages for clinical use.

Is there any advantage to the acquisition of 24-hour thallium images, in the presence of persistent perfusion defects at 4 h after reinjection?

We determined the incidence of delayed 24-h reversibility post thallium-201 reinjection and imaging at 4 h, as well as the prognostic and significance of such delayed reversibility. We studied 46 consecutive patients with persistent thallium-201 perfusion or incompletely reversible single-photon emission tomography (SPET) perfusion defects acquired within 10 min after reinjection performed 4 h after stress. In 38 of 46 patients (82%) 24-h images showed no further reversibility beyond the post-reinjection 4-h study (group A). Eight of 46 patients (17%) demonstrated reversibility on 24-h imaging (group B). Of these eight, three patients showed no improvement compared with the post-stress images, with a mean perfusion score of the abnormal segments of 1. 25+/-0.50 on the 4-h images, and of 3.00 on the 24-h images, where normal is 4. Four patients presented with nine mixed regions. Four of these regions showed an improvement in the mean perfusion score of 2.50+/-0.58 on 4- and 24-h images. Two of them, with moderate/severe defects, demonstrated complete reversibility at 4-h post-reinjection imaging. In addition, five other regions presented no improvement at 4-h imaging, but showed an improvement in the mean perfusion score from 0.80+/-0.84 at 4-h to 3.30+/-0.89 at 24-h imaging. Two of these regions in one patient showed a severe perfusion score of 0 at 4 h, and complete reversibility at 24 hours, with a mean score improvement of 4. Another patient had three severe perfusion defects; two of them redistributed partially at 4 h and completely at 24 h. The remaining segment with a perfusion score of 0 at 4 h, presented complete reversibility with a score of 4 at 24 h. Two (4%) patients revealed significant reversibility at 24 h in a region that was severely underperfused after post-reinjection imaging at 4 h. Among group B patients, 75% (6/8) had recent acute ischemic syndrome, compared with only 13% (5/38) in group A (P = 0. 001). Among 11 patients with unstable angina, six (55%) had evidence of delayed 24-h reversibility, compared with 2 of 35 (6%) patients without clinically acute ischemia (P = 0.001). On follow-up, there were seven (17%) cardiac deaths among the 38 group A patients but three (38%) among the eight group B patients (P = 0.3). These findings suggest that although the presence of delayed 24-h 201Tl, post-reinjection reversibility is infrequent, it has potential clinical importance. Thus, delayed 24-h imaging should be considered in the context of unstable angina or other acute coronary syndromes.

Fast acquisition of myocardial SPECT images with Tc-99m sestamibi for the diagnosis of coronary artery disease.

BACKGROUND: Shortening the acquisition time for myocardial single-photon emission computed tomographic (SPECT) imaging increases patient comfort and laboratory throughput. The purpose of this study was to compare the diagnostic accuracy for coronary artery disease detection of myocardial SPECT images acquired in 5 to 10 minutes versus 25 minutes using Tc-99m methoxyisobutylisonitrile (Tc-99m sestamibi) and a single-head gamma camera. METHODS AND RESULTS: Forty-one subjects had a standard 1-day rest/stress Tc-99m sestamibi myocardial SPECT study. Two sets of rest and stress images were acquired on the same day for each subject. One set of images was acquired with a 5- to 10-minute fast acquisition protocol; the second set of images was acquired with a 25-minute standard protocol. The accuracies of the fast and standard protocols for identifying individuals with and without coronary artery disease were equivalent. Accuracy was 76% for the fast protocol and 73% for the standard protocol in individuals with at least one coronary stenosis > or = 70%. The accuracies of the two protocols for identifying individual coronary arteries with stenoses > or = 70% also were equivalent. Accuracy was 77% for the fast protocol and 74% for the standard protocol. CONCLUSIONS: SPECT myocardial images may be acquired in as little as 5 to 10 minutes using Tc-99m sestamibi and a 1-day rest/stress protocol. Accuracy is equivalent to that attained in studies with longer imaging times.

Strategies for extraction of quantitative data from volumetric dynamic cardiac positron emission tomography data.

The ability of positron emission tomography (PET) to serve as a useful myocardial perfusion indicator is well established. We describe a methodology for obtaining reliable quantitative kinetic parameters from dynamic cardiac PET data. Reconstructed images of the myocardium are subdivided into three-dimensional volumes of interest which are used to obtain quantitative measures of myocardial perfusion over physiologically meaningful anatomical regions. The quantitation technique rigorously models the uncertainty of estimated parameters while compensating for effects such as patient motion and partial volumes to arrive at model parameters with well-established confidence intervals.

Comparison of adenosine to dipyridamole in degree of coronary hyperemic response in heart transplant recipients.

Transplant coronary vasculopathy is associated with endothelial dysfunction. Microvascular function, assessed as coronary flow reserve, has been reported to be normal. We used intracoronary ultrasound technology to simultaneously assess conductance and resistance vessel function in response to standard dosages of the vasodilators adenosine and dipyridamole. Coronary hemodynamic changes were assessed in 11 heart transplant recipients, at a mean duration of 784 +/- 516 days after transplantation, using a 3.2Fr or 4.3Fr, 30-MHz ultrasound imaging catheter over a 0.014-inch Doppler guidewire. Measures of coronary average peak flow velocity (APV) and coronary cross-sectional area (CSA) were used to calculate volumetric flow during intravenous infusions of adenosine (140 micrograms/kg/min over 4 minutes) and dipyridamole (140 micrograms/kg/min over 4 minutes). Flow reserve was assessed as a ratio of maximal pharmacologically induced flow to steady baseline flow before infusion. Increase in APV (261.9% vs 194.6%, p = 0.005), lumenal CSA (+11.8% vs +4.2%, p = 0.01), peak volumetric blood flow (515.8 vs 317.2 ml/min, p = 0.007), and coronary flow reserve (2.93 +/- 0.74 vs 1.99 +/- 0.53, p < 0.001) were higher with adenosine than dipyridamole. Both agents caused similar decreases in systemic blood pressure and little change in heart rate. Adenosine appears to be a more potent coronary vasodilator than dipyridamole in denervated human transplant subjects. Adenosine has a vasodilator effect at the epicardial and microvascular levels, resulting in an overall increase in volumetric flow. Flow reserve in response to both endothelium-independent agents is decreased in comparison with previously established values, but the attenuation is greater with dipyridamole.

Diagnostic utility of tomographic myocardial perfusion imaging with technetium 99m furifosmin (Q12) compared with thallium 201: results of a phase III multicenter trial.

BACKGROUND: Based on physical properties, 99mTc-labeled perfusion agents offer several advantages over 201Tl for myocardial perfusion imaging. The results of in vivo and experimental studies, along with preliminary experience in human subjects, have shown 99mTc-labeled furifosmin to be a promising new perfusion tracer. The purpose of this study was to evaluate the safety of a new myocardial perfusion agent, 99mTc-labeled furifosmin (Q12), and determine the concordance of furifosmin perfusion scintigraphy to 201Tl imaging. In addition, we sought to determine the normalcy rate of myocardial scintigraphy with furifosmin. METHODS AND RESULTS: One hundred fifty patients constituted the study group in this multicenter trial. Patients underwent exercise testing with furifosmin injected at peak exercise, and tomographic imaging was begun 15 to 30 minutes afterward. After a separate injection, resting images were obtained 3 to 4 hours later. Thallium scintigraphy was performed within 2 weeks of the furifosmin scans, after a similar exercise workload. Patients with a low likelihood of coronary artery disease (n = 39) also underwent furifosmin imaging. All images were processed and displayed in uniform manner and interpreted by a panel of readers. No adverse effects or clinically important laboratory alterations were related to furifosmin imaging. Image quality was slightly better with furifosmin than with thallium. The overall concordance between the perfusion studies was 86% (kappa value = 0.669). The normalcy rate for furifosmin scintigraphy was 100%. CONCLUSIONS: 99mTc-labeled furifosmin is a promising new 99mTc-labeled myocardial perfusion agent, providing diagnostic results similar to those obtained with 201Tl.

Cardiac activation mapping by MRI.

To establish cardiac MRI as a tool for noninvasive evaluation of activation patterns, 10 healthy volunteers were examined by cine segmented turboFLASH imaging sequences. Sequence modifications for low signal blood-pool appearance were applied, i.e., bilateral spatial saturation for segmented turboFLASH imaging. Pixelwise calculation of first-harmonic Fourier phase values (displayed as color-encoded maps) reveal either anterior septal or left ventricular free-wall sites as areas of earliest phase spreading towards posterior paraseptal sites in segmented turboFLASH scans. Phase scatter is lower in unsaturated than spatially presaturated segmented turboFLASH studies. Phase standard deviation in areas of endocardial displacement is higher in basal than apical slice positions in these scans. Early results indicate that first-harmonic Fourier phase analysis of cardiac-segmented turboFLASH MRI cine studies may provide a tool for noninvasive studies of cardiac activation sequence.

Stress imaging. Current clinical options for the diagnosis, localization, and evaluation of coronary artery disease.

As technology advances, new methods evolve. In this article, the methods of stress testing and related imaging in coronary disease are addressed, and dynamic and pharmacologic stress, direct and indirect methods, are defined and evaluated. The stress imaging methods related to the modalities of scintigraphy and ultrasound are reviewed and their advantages and disadvantages assessed in view of scientific and economic factors.