Retrospective Analysis of Contrast-enhanced Ultrasonography Effectiveness in Reducing Time to Diagnosis and Imaging-related Expenditures at a Single Large United States County Hospital.

Hepatic and renal lesions detected during ultrasound examinations frequently require subsequent contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) for characterization, delaying time to imaging diagnosis and increasing overall health care expenditures. Contrast-enhanced ultrasonography (CEUS) is a comparatively low-cost diagnostic tool that is underutilized in the evaluation of such indeterminate or suspicious hepatic and renal lesions. A retrospective chart review of CEUS examinations performed in our department demonstrated significantly shorter time to imaging diagnosis with CEUS compared to CT or MRI, largely due to the ability to perform the CEUS examination at the time of initial examination. For example mean time to completion for outpatient examinations was 5.2, 52.3, and 123.5 days for CEUS, CT, and MRI, respectively. The majority (78.4%) of CEUS examinations were completed the same day as the initial examination. Additionally, 66.7% of CEUS examinations were deemed diagnostic, abrogating further workup with CT or MRI in most cases. Annual imaging cost reduction of up to US $117,000 is anticipated in our institution based on projected reductions in follow-up CT and MRI examinations. These results indicate when CEUS was used as a first step to characterize both incidental lesions in patients without known risk factors for malignancy as well as suspicious lesions in patients with risk factors it can greatly reduce time to diagnosis and health care expenditures.

REST regulates the pool size of the different neural lineages by restricting the generation of neurons and oligodendrocytes from neural stem/progenitor cells.

REST is a master repressor of neuronal genes; however, whether it has any role during nervous system development remains largely unknown. Here, we analyzed systematically the role of REST in embryonic stem cells and multipotent neural stem/progenitor (NS/P) cells, including neurogenic and gliogenic NS/P cells derived from embryonic stem (ES) cells or developing mouse embryos. We showed that REST-null ES cells remained pluripotent and generated teratomas consisting of the three germ layers. By contrast, multipotent NS/P cells lacking REST displayed significantly reduced self-renewal capacity owing to reduced cell cycle kinetics and precocious neuronal differentiation. Importantly, although early-born neurogenic NS/P cells that lack REST were capable of differentiating to neurons and glia, the neuronal and oligodendrocytic pools were significantly enlarged and the astrocytic pool was shrunken. However, gliogenic NS/P cells lacking REST were able to generate a normal astrocytic pool size, suggesting that the shrinkage of the astrocytic pool generated from neurogenic NS/P cells lacking REST probably occurs by default. Microarray profiling of early-born NS/P cells lacking REST showed upregulation of neuronal as well as oligodendrocytic genes, specifically those involved in myelination. Furthermore, chromatin immunoprecipitation analyses showed that some of the upregulated oligodendrocytic genes contain an RE1 motif and are direct REST targets. Together, our data support a central role for REST during neural development in promoting NS/P cell self-renewal while restricting the generation and maturation of neurons and oligodendrocytes.

Retinoid-induced expression and activity of an immediate early tumor suppressor gene in vascular smooth muscle cells.

Retinoids are used clinically to treat a number of hyper-proliferative disorders and have been shown in experimental animals to attenuate vascular occlusive diseases, presumably through nuclear receptors bound to retinoic acid response elements (RARE) located in target genes. Here, we show that natural or synthetic retinoids rapidly induce mRNA and protein expression of a specific isoform of A-Kinase Anchoring Protein 12 (AKAP12ß) in cultured smooth muscle cells (SMC) as well as the intact vessel wall. Expression kinetics and actinomycin D studies indicate Akap12ß is a retinoid-induced, immediate-early gene. Akap12ß promoter analyses reveal a conserved RARE mildly induced with atRA in a region that exhibits hyper-acetylation. Immunofluorescence microscopy and protein kinase A (PKA) regulatory subunit overlay assays in SMC suggest a physical association between AKAP12ß and PKA following retinoid treatment. Consistent with its designation as a tumor suppressor, inducible expression of AKAP12ß attenuates SMC growth in vitro. Further, immunohistochemistry studies establish marked decreases in AKAP12 expression in experimentally-injured vessels of mice as well as atheromatous lesions in humans. Collectively, these results demonstrate a novel role for retinoids in the induction of an AKAP tumor suppressor that blocks vascular SMC growth thus providing new molecular insight into how retiniods may exert their anti-proliferative effects in the injured vessel wall.

SRF and myocardin regulate LRP-mediated amyloid-beta clearance in brain vascular cells.

Amyloid beta-peptide (Abeta) deposition in cerebral vessels contributes to cerebral amyloid angiopathy (CAA) in Alzheimer's disease (AD). Here, we report that in AD patients and two mouse models of AD, overexpression of serum response factor (SRF) and myocardin (MYOCD) in cerebral vascular smooth muscle cells (VSMCs) generates an Abeta non-clearing VSMC phenotype through transactivation of sterol regulatory element binding protein-2, which downregulates low density lipoprotein receptor-related protein-1, a key Abeta clearance receptor. Hypoxia stimulated SRF/MYOCD expression in human cerebral VSMCs and in animal models of AD. We suggest that SRF and MYOCD function as a transcriptional switch, controlling Abeta cerebrovascular clearance and progression of AD.

Overexpression of A-kinase anchoring protein 12A activates sterol regulatory element binding protein-2 and enhances cholesterol efflux in hepatic cells.

A-kinase anchoring protein 12 (AKAP12) is known to function as a scaffold protein and as a putative tumor suppressor. However, little is known about the biological role of AKAP12 in hepatic cells. In this study, we performed micro-array analysis to identify the downstream pathway of AKAP12A, and found that AKAP12A overexpression up-regulates the expressions of several cholesterol-associated genes including HMG-CoA reductase and LDL receptor, which have been reported to be controlled by sterol regulatory element binding protein-2 (SREBP-2). It was found that AKAP12A activates SREBP-2 in hepatic cells, as demonstrated by the presence of its cleavage product, whereas the activation of sterol regulatory element binding protein-1 was not remarkably changed. Moreover, AKAP12A-induced SREBP-2 activation was found to depend on SREBP cleavage-activating protein (SCAP), as inhibition of SCAP by RNAi or sterols blocked SREBP-2 activation in response to AKAP12A overexpression. Interestingly, the hydrophobic amine U18666A caused dramatic movement of AKAP12A from the plasma membrane to cytosol and lysosomal membranes. Moreover, cholesterol depletion from the plasma membrane (using methyl-beta-cyclodextrin) caused a shift of AKAP12A from the plasma membrane to the cytoplasm. Cholesterol binding assay revealed that the N-terminal region of AKAP12A binds directly to cholesterol in vitro. Furthermore, AKAP12A overexpression enhanced [3H]-cholesterol efflux to extracellular acceptors, suggesting that AKAP12A may activate SREBP-2 by increasing cholesterol efflux. In conclusion, the present study suggests that AKAP12A is a novel regulator of cellular cholesterol metabolism.

Serum response factor and myocardin mediate arterial hypercontractility and cerebral blood flow dysregulation in Alzheimer's phenotype.

Cerebral angiopathy contributes to cognitive decline and dementia in Alzheimer's disease (AD) through cerebral blood flow (CBF) reductions and dysregulation. We report vascular smooth muscle cells (VSMC) in small pial and intracerebral arteries, which are critical for CBF regulation, express in AD high levels of serum response factor (SRF) and myocardin (MYOCD), two interacting transcription factors that orchestrate a VSMC-differentiated phenotype. Consistent with this finding, AD VSMC overexpressed several SRF-MYOCD-regulated contractile proteins and exhibited a hypercontractile phenotype. MYOCD overexpression in control human cerebral VSMC induced an AD-like hypercontractile phenotype and diminished both endothelial-dependent and -independent relaxation in the mouse aorta ex vivo. In contrast, silencing SRF normalized contractile protein content and reversed a hypercontractile phenotype in AD VSMC. MYOCD in vivo gene transfer to mouse pial arteries increased contractile protein content and diminished CBF responses produced by brain activation in wild-type mice and in two AD models, the Dutch/Iowa/Swedish triple mutant human amyloid beta-peptide (Abeta)-precursor protein (APP)- expressing mice and APPsw(+/-) mice. Silencing Srf had the opposite effect. Expression of SRF did not change in VSMC subjected to Alzheimer's neurotoxin, Abeta. Thus, SRF-MYOCD overexpression in small cerebral arteries appears to initiate independently of Abeta a pathogenic pathway mediating arterial hypercontractility and CBF dysregulation, which are associated with Alzheimer's dementia.

Tissue expression of the novel serine carboxypeptidase Scpep1.

We previously identified a novel gene designated retinoid-inducible serine carboxypeptidase (RISC or Scpep1). Here we characterize a polyclonal antibody raised to Scpep1 and assess its localization in mouse cells and tissues. Western blot analysis revealed an immunospecific approximately 35-kDa protein corresponding to endogenous Scpep1. This protein is smaller than the predicted approximately 51-kDa, suggesting that Scpep1 is proteolytically cleaved to a mature enzyme. Immunohistochemical studies demonstrate Scpep1 expression in embryonic heart and vasculature as well as in adult aortic smooth muscle cells and endothelial cells. Scpep1 displays a broad expression pattern in adult tissues with detectable levels in epithelia of digestive tract and urinary bladder, islet of Langerhans, type II alveolar cells and macrophages of lung, macrophage-like cells of lymph nodes and spleen, Leydig cells of testis, and nerve fibers in brain and ganglia. Consistent with previous mRNA studies in kidney, Scpep1 protein is restricted to proximal convoluted tubular epithelium (PCT). Immunoelectron microscopy shows enriched Scpep1 within lysosomes of the PCT, and immunofluorescence microscopy colocalizes Scpep1 with lysosomal-associated membrane protein-2. These results suggest that Scpep1 is a widely distributed lysosomal protease requiring proteolytic cleavage for activity. The highly specific Scpep1 antibody characterized herein provides a necessary reagent for elucidating Scpep1 function.

Defining the mammalian CArGome.

Serum response factor (SRF) binds a 1216-fold degenerate cis element known as the CArG box. CArG boxes are found primarily in muscle- and growth-factor-associated genes although the full spectrum of functional CArG elements in the genome (the CArGome) has yet to be defined. Here we describe a genome-wide screen to further define the functional mammalian CArGome. A computational approach involving comparative genomic analyses of human and mouse orthologous genes uncovered >100 hypothetical SRF-dependent genes, including 10 previously identified SRF targets, harboring a conserved CArG element within 4000 bp of the annotated transcription start site (TSS). We PCR-cloned 89 hypothetical SRF targets and subjected each of them to at least two of several validations including luciferase reporter, gel shift, chromatin immunoprecipitation, and mRNA expression following RNAi knockdown of SRF; 60/89 (67%) of the targets were validated. Interestingly, 26 of the validated SRF target genes encode for cytoskeletal/contractile or adhesion proteins. RNAi knockdown of SRF diminishes expression of several SRF-dependent cytoskeletal genes and elicits an attending perturbation in the cytoarchitecture of both human and rodent cells. These data illustrate the power of integrating existing algorithms to interrogate the genome in a relatively unbiased fashion for cis-regulatory element discovery. In this manner, we have further expanded the mammalian CArGome with the discovery of an array of cyto-contractile genes that coordinate normal cytoskeletal homeostasis. We suggest one function of SRF is that of an ancient master regulator of the actin cytoskeleton.

Cross-species sequence analysis reveals multiple charged residue-rich domains that regulate nuclear/cytoplasmic partitioning and membrane localization of a kinase anchoring protein 12 (SSeCKS/Gravin).

A kinase anchoring proteins (AKAPs) assemble and compartmentalize multiprotein signaling complexes at discrete subcellular locales and thus confer specificity to transduction cascades using ubiquitous signaling enzymes, such as protein kinase A. Intrinsic targeting domains in each AKAP determine the subcellular localization of these complexes and, along with protein-protein interaction domains, form the core of AKAP function. As a foundational step toward elucidating the relationship between location and function, we have used cross-species sequence analysis and deletion mapping to facilitate the identification of the targeting determinants of AKAP12 (also known as SSeCKS or Gravin). Three charged residue-rich regions were identified that regulate two aspects of AKAP12 localization, nuclear/cytoplasmic partitioning and perinuclear/cell periphery targeting. Using deletion mapping and green fluorescent protein chimeras, we uncovered a heretofore unrecognized nuclear localization potential. Five nuclear localization signals, including a novel class of this type of signal termed X2-NLS, are found in the central region of AKAP12 and are important for nuclear targeting. However, this nuclear localization is suppressed by the negatively charged C terminus that mediates nuclear exclusion. In this condition, the distribution of AKAP12 is regulated by an N-terminal targeting domain that simultaneously directs perinuclear and peripheral AKAP12 localization. Three basic residue-rich regions in the N-terminal targeting region have similarity to the MARCKS proteins and were found to control AKAP12 localization to ganglioside-rich regions at the cell periphery. Our data suggest that AKAP12 localization is regulated by a hierarchy of targeting domains and that the localization of AKAP12-assembled signaling complexes may be dynamically regulated.

AKAP12alpha, an atypical serum response factor-dependent target gene.

We recently identified three AKAP12 isoforms that are differentially regulated by distinct promoters. During a screen to identify molecular determinants distinguishing the activities of these promoters, we found a potential binding site for the serum response factor (SRF) in the promoter of the ubiquitously expressed AKAP12alpha isoform. SRF is an evolutionarily conserved transcription factor that governs disparate programs of gene expression linked to cellular growth and differentiation. Using a combination of reporter assays and RNA interference, we demonstrate that SRF is required for AKAP12alpha expression. SRF regulates the activity of the AKAP12alpha promoter through two conserved CArG boxes that bind SRF with different affinities. Unlike other SRF-dependent genes, AKAP12alpha is not regulated by growth or differentiation stimuli. Molecular analysis of the AKAP12alpha SRF-binding sites, or CArG boxes, indicates that sequences flanking these sites are the determinants of sensitivity to SRF-activating signals. Specifically, the AKAP12alpha CArG boxes are shielded from growth stimulation by the absence of a binding site for Ets transcription factors. Similarly, sensitivity to the differentiation-associated co-factor, myocardin, was also determined by responsive flanking sequence; however, unlike growth stimuli, sensitivity to myocardin was found to also be dependent on a consensus CArG box. Collectively, our data demonstrate that AKAP12alpha belongs to a novel class of atypical SRF-dependent target genes. Furthermore, we provide new insight into the role of flanking sequences in determining sensitivity to SRF-myocardin activity.

Multiple promoters direct expression of three AKAP12 isoforms with distinct subcellular and tissue distribution profiles.

A Kinase Anchoring Protein 12 (AKAP12; also known as src-suppressed C kinase substrate (SSeCKS) and Gravin) is a multivalent anchoring protein with tumor suppressor activity. Although expression of AKAP12 has been examined in a number of contexts, its expression control remains to be elucidated. Herein, we characterize the genomic organization of the AKAP12 locus, its regulatory regions, and the spatial distribution of the proteins encoded by the AKAP12 gene. Using comparative genomics and various wet-lab assays, we show that the AKAP12 locus is organized as three separate transcription units that are governed by non-redundant promoters coordinating distinct tissue expression profiles. The proteins encoded by the three AKAP12 isoforms (designated alpha, beta, and gamma) share >95% amino acid sequence identity but differ at their N termini. Analysis of the targeting of each isoform reveals distinct spatial distribution profiles. An N-terminal myristoylation motif present in AKAP12alpha is shown to be necessary and sufficient for targeted expression of this AKAP12 isoform to the endoplasmic reticulum, a novel subcellular compartment for AKAP12. Our results demonstrate heretofore unrecognized complexity within the AKAP12 locus and suggest a mechanism for genetic control of signaling specificity through distinct regulation of alternately targeted anchoring protein isoforms.

Thioredoxin-2 inhibits mitochondria-located ASK1-mediated apoptosis in a JNK-independent manner.

Apoptosis signal-regulating kinase 1 (ASK1) mediates cytokines and oxidative stress (ROS)-induced apoptosis in a mitochondria-dependent pathway. However, the underlying mechanism has not been defined. In this study, we show that ASK1 is localized in both cytoplasm and mitochondria of endothelial cells (ECs) where it binds to cytosolic (Trx1) and mitochondrial thioredoxin (Trx2), respectively. Cys-250 and Cys-30 in the N-terminal domain of ASK1 are critical for binding of Trx1 and Trx2, respectively. Mutation of ASK1 at C250 enhanced ASK1-induced JNK activation and apoptosis, whereas mutation of ASK1 at C30 specifically increased ASK1-induced apoptosis without effects on JNK activation. We further show that a JNK-specific inhibitor SP600125 completely blocks TNF induced JNK activation, Bid cleavage, and Bax mitochondrial translocation, but only partially inhibits cytochrome c release and EC death, suggesting that TNF induces both JNK-dependent and JNK-independent apoptotic pathways in EC. Mitochondria-specific expression of a constitutively active ASK1 strongly induces EC apoptosis without JNK activation, Bid cleavage, and Bax mitochondrial translocation. These data suggest that mitochondrial ASK1 mediates a JNK-independent apoptotic pathway induced by TNF. To determine the role of Trx2 in regulation of mitochondrial ASK1 activity, we show that overexpression of Trx2 inhibits ASK1-induced apoptosis without effects on ASK1-induced JNK activation. Moreover, specific knockdown of Trx2 in EC increases TNF/ASK1-induced cytochrome c release and cell death without increase in JNK activation, Bid cleavage, and Bax translocation. Our data suggest that ASK1 in cytoplasm and mitochondria mediate distinct apoptotic pathways induced by TNF, and Trx1 and Trx2 cooperatively inhibit ASK1 activities.

Retinoids: pleiotropic agents of therapy for vascular diseases?

Retinoids, the natural and synthetic derivatives of vitamin A, exert broad biological effects and have been used clinically to treat a variety of dermatological and neoplastic diseases. The principal mode of action of many retinoids is through the binding and activation of a family of nuclear receptors that modulate gene transcription. Recent evidence demonstrates that retinoids effectively attenuate experimental vessel wall narrowing due to atherosclerosis, post-balloon injury stenosis, and bypass graft failure. Moreover, retinoids promote a differentiated phenotype in smooth muscle cells (SMC) which, unlike other muscle types, is not fixed and is subject to considerable modulation in disease states. A growing number of in vitro studies have reported desirable effects of retinoids on cell migration, proliferation, apoptosis, matrix remodeling, fibrinolysis, coagulation, and inflammation, all of which impinge on vascular disease. Since vascular SMC and endothelial cells (EC) express most retinoid receptors, the mechanisms underlying retinoid-mediated events in these cells and the vessel wall likely relate to an altered transcriptome. In fact, there is a growing list of retinoid-response genes encoding proteins that likely mediate the actions of retinoids. Retinoid-response genes, therefore, represent promising targets of therapy for the refined treatment of vascular diseases. The purpose of this review is to summarize the emerging importance of retinoids in the control of vascular cell responses with special emphasis on potential mechanisms underlying retinoid-induced changes in the vessel wall following injury. Given the similarities in the pathogenesis of neoplasia and vascular disease, it is reasonable to consider testing the efficacy of retinoids for the treatment of human vascular disease.

Myocardin: a component of a molecular switch for smooth muscle differentiation.

A hallmark of smooth muscle cells (SMCs) in culture and the injured vessel wall is their phenotypic modulation from a differentiated state to one of heightened growth, migration, and matrix synthesis. The transcriptional mechanisms underlying this altered genetic program have yet to be elucidated. Serum response factor (SRF) has emerged as a critical regulator of SMC-restricted gene expression via its interaction with proximal CArG elements; however, levels of SRF protein do not change during SMC phenotypic modulation, suggesting a role for other factors or events in this process. One such factor could be myocardin, a novel SRF coactivator recently cloned from cardiac tissue. Levels of myocardin are abundantly expressed in rat aortic media along with key SMC-restricted genes. In several SMC lines, myocardin mRNA levels decrease in parallel with the loss or attenuation of SMC marker expression. Transient transfection experiments with CMV-driven myocardin in both SMC and non-SMC reveal CArG-dependent transactivation of the SM-Calp promoter-enhancer. Several additional CArG-dependent SMC promoters show variable activation in a cell-and promoter-context dependent manner. To determine whether myocardin could activate an endogenous program of SMC differentiation, we stably transfected L6 myoblasts and assessed SMC marker expression and growth. Results reveal the expression of several SMC markers concomitant with a lower growth potential. Collectively, these studies suggest that myocardin is an important component of a molecular switch for the SMC differentiation program.