Smooth muscle cell (SMC) proliferation and migration are substantially controlled by the platelet-derived growth factor receptor-␤ (PDGFR␤), which can be regulated by the Ser/Thr kinase G protein-coupled receptor kinase-2 (GRK2). In mouse aortic SMCs, however, we found that prolonged PDGFR␤ activation engendered down-regulation of GRK5, but not GRK2; moreover, GRK5 and PDGFR␤ were coordinately up-regulated in SMCs from atherosclerotic arteries. With SMCs from GRK5 knock-out and cognate wild type mice (five of each), we found that physiologic expression of GRK5 increased PDGF-promoted PDGFR␤ seryl phosphorylation by 3-fold and reduced PDGFR␤-promoted phosphoinositide hydrolysis, thymidine incorporation, and overall PDGFR␤ tyrosyl phosphorylation by ϳ35%. Physiologic SMC GRK5 activity also increased PDGFR␤ association with the phosphatase Shp2 (8-fold), enhanced phosphorylation of PDGFR␤ Tyr 1009 (the docking site for Shp2), and reduced phosphorylation of PDGFR␤ Tyr 1021. Consistent with having increased PDGFR␤-associated Shp2 activity, GRK5-expressing SMCs demonstrated greater PDGF-induced Src activation than GRK5-null cells. GRK5-mediated desensitization of PDGFR␤ inositol phosphate signaling was diminished by Shp2 knock-down or impairment of PDGFR␤/Shp2 association. In contrast to GRK5, physiologic GRK2 activity did not alter PDGFR␤/Shp2 association. Finally, purified GRK5 effected agonist-dependent seryl phosphorylation of partially purified PDGFR␤s. We conclude that GRK5 mediates the preponderance of PDGF-promoted seryl phosphorylation of the PDGFR␤ in SMCs, and, through mechanisms involving Shp2, desensitizes PDGFR␤ inositol phosphate signaling and enhances PDGFR␤-triggered Src activation.

Acoustic Radiation Force (ARF)-based methods have been demonstrated to be a viable tool for noninvasively estimating tissue elastic properties, and shear wave velocimetry has been used to quantitatively measure the stiffening and relaxation of myocardial tissue in open-chest experiments. Dynamic stiffness metrics may prove to be indicators for certain cardiac diseases, but a clinically-viable means of remotely generating and tracking transverse wave propagation in myocardium is needed. Intracardiac echocardiography (ICE) catheter-tip transducers are demonstrated here as a viable tool for making this measurement. ICE probes achieve favorable proximity to the myocardium, enabling the use of shear wave velocimetry from within the right ventricle throughout the cardiac cycle. This work describes the techniques used to overcome the challenges of using a small probe to perform ARF-driven shear wave velocimetry, and presents in vivo porcine data showing the effectiveness of this method in the interventricular septum. Acoustic Radiation Force (ARF)-based methods have been demonstrated to be a viable tool for noninvasively estimating tissue elastic properties, and shear wave velocimetry has been used to quantitatively measure the stiffening and relaxation of myocardial tissue in open-chest experiments. Dynamic stiffness metrics may prove to be indicators for certain cardiac diseases, but a clinically-viable means of remotely generating and tracking transverse wave propagation in myocardium is needed. Intracardiac echocardiography (ICE) catheter-tip transducers are demonstrated here as a viable tool for making this measurement. ICE probes achieve favorable proximity to the myocardium, enabling the use of shear wave velocimetry from within the right ventricle throughout the cardiac cycle. This work describes the techniques used to overcome the challenges of using a small probe to perform ARF-driven shear wave velocimetry, and presents in vivo porcine data showing the effectiveness of this method in the interventricular septum.

G protein-coupled receptor kinase-2 (GRK2) serinephosphorylates the platelet-derived growth factor receptor-␤ (PDGFR␤), and thereby diminishes signaling by the receptor. Because activation of GRK2 may involve phosphorylation of its N-terminal tyrosines by cSrc, we tested whether the PDGFR␤ itself could tyrosine-phosphorylate and activate GRK2. To do so, we used wild type (WT) and Y857F mutant PDGFR␤s in HEK cells, which lack endogenous PDGFRs. The Y857F PDGFR␤ autophosphorylates normally but does not phosphorylate exogenous substrates. Although PDGFstimulated Y857F and WT PDGFR␤s activated c-Src equivalently, the WT PDGFR␤ tyrosine-phosphorylated GKR2 60-fold more than the Y857F PDGFR␤ in intact cells. With purified GRK2 and either WT or Y857F PDGFR␤s immunoprecipitated from HEK cells, GRK2 tyrosyl phosphorylation was PDGF-dependent and required the WT PDGFR␤, even though the WT and Y857F PDGFR␤s autophosphorylated equivalently. This PDGFR␤-mediated GRK2 tyrosyl phosphorylation enhanced GRK2 activity: GRK2-mediated seryl phosphorylation of the PDGFR␤ was 9-fold greater for the WT than for the Y857F in response to PDGF, but equivalent when GRK2 was activated by sequential stimulation of ␤ 2 -adrenergic and PDGF-␤ receptors. Furthermore, both PDGFR␤-mediated GRK2 tyrosyl phosphorylation and GRK2-mediated PDGFR␤ seryl phosphorylation were reduced ϳ50% in intact cells by mutation to phenylalanine of three tyrosines in the N-terminal domain of GRK2. We conclude that the activated PDGFR␤ itself phosphorylates GRK2 tyrosyl residues and thereby activates GRK2, which then serine-phosphorylates and desensitizes the PDGFR␤.As a receptor protein-tyrosine kinase, the platelet-derived growth factor receptor-␤ (PDGFR␤) 1 triggers cellular proliferation, migration, and survival (1) but also contributes to atherosclerosis (2-4) and malignant neoplasia (5, 6). Agonist-induced dimerization of the PDGFR␤ enables receptor activation consequent to autophosphorylation (7), followed by recruitment to the PDGFR␤ of various signaling proteins, and tyrosyl phosphorylation of PDGFR␤ substrates (1). In order for the PDGFR␤ to phosphorylate these "exogenous" substrates, however, the PDGFR␤ must be autophosphorylated on Tyr 857 , located in the PDGFR␤ kinase activation loop (8).Regulatory constraints on PDGFR␤ signaling include tyrosyl dephosphorylation (9, 10), degradation and down-regulation of cellular PDGFR␤s (11, 12), and agonist-induced phosphorylation of the PDGFR␤ on serine residues (13-15). In fibroblasts, the preponderance of this PDGFR␤ seryl phosphorylation appears to be mediated by GRK2 (13), a ubiquitous allosteric kinase that also phosphorylates activated G protein-coupled (heptahelical) receptors and thereby initiates their desensitization (16). We have demonstrated GRK2-mediated PDGFR␤ seryl phosphorylation with purified kinase preparations (14), as well as by comparing PDGFR␤ seryl phosphorylation in GRK2-null, cognate WT, and GRK2 "add-back" fibroblasts (13). GRK2-mediated PDGFR␤ phosphorylation diminishes PDGFR␤ tyrosyl ph...

Short-Lag Spatial Coherence (SLSC) imaging is a novel beamforming technique that reduces acoustic clutter in ultrasound images. A clinical study was conducted to investigate clutter reduction and endocardial border detection in cardiac SLSC images. Individual channel echo data were acquired from the left ventricle of 14 volunteers, after informed consent and IRB approval. Paired B-mode and SLSC images were created from these data. Contrast, contrast-to-noise, and signal-to-noise ratios were measured in paired images, and these metrics were improved with SLSC imaging in most cases. Three cardiology fellows rated the visibility of endocardial segments in randomly ordered B-mode and SLSC cine loops. SLSC imaging offered 22–33% improvement (p < 0.05) in endocardial border visibility when B-mode image quality was poor (i.e. 80% or more of the endocardial segments could not be visualized by the three reviewers). The percentage of volunteers with poor-quality images was decreased from 21% to 7% with the SLSC beamformer. Results suggest that SLSC imaging has the potential to improve clinical cardiac assessments that are challenged by clutter.

BackgroundArtificial intelligence (AI) techniques are increasingly applied to cardiovascular (CV) medicine in arenas ranging from genomics to cardiac imaging analysis. Cardiac Phase Space Tomography Analysis (cPSTA), employing machine-learned linear models from an elastic net method optimized by a genetic algorithm, analyzes thoracic phase signals to identify unique mathematical and tomographic features associated with the presence of flow-limiting coronary artery disease (CAD). This novel approach does not require radiation, contrast media, exercise, or pharmacological stress. The objective of this trial was to determine the diagnostic performance of cPSTA in assessing CAD in patients presenting with chest pain who had been referred by their physician for coronary angiography.MethodsThis prospective, multicenter, non-significant risk study was designed to: 1) develop machine-learned algorithms to assess the presence of CAD (defined as one or more ≥ 70% stenosis, or fractional flow reserve ≤ 0.80) and 2) test the accuracy of these algorithms prospectively in a naïve verification cohort. This report is an analysis of phase signals acquired from 606 subjects at rest just prior to angiography. From the collective phase signal data, features were extracted and paired with the known angiographic results. A development set, consisting of signals from 512 subjects, was used for machine learning to determine an algorithm that correlated with significant CAD. Verification testing of the algorithm was performed utilizing previously untested phase signals from 94 subjects.ResultsThe machine-learned algorithm had a sensitivity of 92% (95% CI: 74%-100%) and specificity of 62% (95% CI: 51%-74%) on blind testing in the verification cohort. The negative predictive value (NPV) was 96% (95% CI: 85%-100%).ConclusionsThese initial multicenter results suggest that resting cPSTA may have comparable diagnostic utility to functional tests currently used to assess CAD without requiring cardiac stress (exercise or pharmacological) or exposure of the patient to radioactivity.

Four pigs, three with focal infarctions in the apical intraventricular septum (IVS) and/or left ventricular free wall (LVFW), were imaged with an intracardiac echocardiography (ICE) transducer. Custom beam sequences were used to excite the myocardium with focused acoustic radiation force (ARF) impulses and image the subsequent tissue response. Tissue displacement in response to the ARF excitation was calculated with a phase-based estimator, and transverse wave magnitude and velocity were each estimated at every depth. The excitation sequence was repeated rapidly, either in the same location to generate 40 Hz M-Modes at a single steering angle, or with a modulated steering angle to synthesize 2-D displacement magnitude and shear wave velocity images at 17 points in the cardiac cycle. Both types of images were acquired from various views in the right and left ventricles, in and out of infarcted regions. In all animals, ARFI and SWEI estimates indicated diastolic relaxation and systolic contraction in non-infarcted tissues. The M-Mode sequences showed high beat-to-beat spatio-temporal repeatability of the measurements for each imaging plane. In views of noninfarcted tissue in the diseased animals, no significant elastic remodeling was indicated when compared to the control. Where available, views of infarcted tissue were compared to similar views from the control animal. In views of the LVFW, the infarcted tissue presented as stiff and non-contractile compared to the control. In a view of the IVS, no significant difference was seen between infarcted and healthy tissue, while in another view, a heterogeneous infarction was seen presenting itself as non-contractile in systole.

Clutter, a problematic noise artifact in echocardiography, appears as a diffuse haze that obscures endocardial borders and inhibits accurate diagnoses. Several approaches are available to reduce clutter in cardiac images, yet difficult-to-image patients still exist. We have recently developed a novel imaging method, termed short-lag spatial coherence (SLSC) imaging, that has demonstrated potential to reduce clutter in simulated and experimental data. With this technique, images are created from the same individual channel signals used to form B-mode images, but instead of applying a conventional delayand-sum beamformer, the data are cross-correlated to measure and display differences in spatial coherence. This technique was applied to in vivo cardiac images. Individual channel signals were acquired to form matched B-mode and SLSC images of the left ventricle in fourteen human volunteers. The contrast and contrast-to-noise ratio (CNR) of the ventricle and the signal-to-noise ratio (SNR) of the endocardium were measured in the same locations in matched B-mode and SLSC images. In SLSC images created with a short-lag value equivalent to 16% of the transmit aperture, contrast and CNR was improved by 9±7 dB and 0.4±0.2, respectively, in the SLSC images. The average SNR of the endocardium was 1.7±0.4 in the SLSC images and 1.8±0.4 in the B-mode images. The presented approach demonstrates a new method for reducing clutter in cardiac images.

Endocardial border visualization, a common task in echochardiography, is typically challenged by the presence of acoustic clutter. This study investigates endocardial border visibility in co-registered fundamental and harmonic data when utilizing the Short-Lag Spatial Coherence (SLSC) beamformer, a clutter reduction approach that we developed. Individual channel echo data were acquired from the left ventricle of 12 volunteers, after informed consent and IRB approval, to create matched image quadruplets of fundamental and harmonic B-mode and SLSC images. Contrast-to-noise ratios (CNR) were measured, and three cardiologists rated the visibility of endocardial segments in randomly ordered cine loops. The statistical significance of visibility ratings was determined with a Holm-Bonferroni correction at a significance level, α = 0.05. CNR increased approximately twofold in fundamental and harmonic SLSC images compared to fundamental and harmonic B-mode images. Fundamental and Harmonic SLSC imaging offered the greatest benefits when fundamental B-mode image quality was poor. Improvements in endocardial segment visibility in short-axis views ranged from 16-28% (α = 0.05) compared to fundamental B-mode images, while improvements in the apical four chamber views ranged from 22-35% (α = 0.05) compared to fundamental and harmonic B-mode images. Results suggest that SLSC and HSCI have the potential to increase endocardial border visualization and thereby improve cardiac assessments of poor-quality B-mode images.