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.