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Biomedical subjects

K Walsh

Publications and source records attributed to K Walsh.

At least 145 records · Page 8Linked to original sources
PubMed1997

Adenoviral constructs encoding phosphorylation-competent full-length and truncated forms of the human retinoblastoma protein inhibit myocyte proliferation and neointima formation.

BACKGROUND: The retinoblastoma (Rb) protein is a key cell-cycle regulator that controls entry into the S phase by modulating the activity of the E2F transcription factor. We analyzed the effects of full-length phosphorylation-competent and a mutant truncated form of human Rb for their effects on vascular smooth muscle cell (VSMC) proliferation and neointima formation. METHODS AND RESULTS: A number of mutant forms, both phosphorylation competent and incompetent, of human Rb protein were evaluated for their ability to inhibit E2F activity. The results of these assays indicated that a phosphorylation competent, amino-terminal-truncated Rb protein (Rb56) was a particularly potent inhibitor of E2F-mediated transcription relative to the full-length Rb construct (Rb110). Adenoviral constructs containing Rb56 or Rb110 expressed their respective Rb forms in VSMCs, as determined by Western immunoblot analysis, and were similar in their abilities to arrest the cell cycle, as determined by assays of 3H-thymidine incorporation and by flow cytometric analyses. When examined for their effect on neointima formation after balloon injury of the rat carotid artery, both full-length and truncated forms of Rb inhibited formation of this VSMC-derived lesion. CONCLUSIONS: These analyses revealed that the maintenance of high levels of phosphorylation-competent human Rb, either full-length or truncated forms, in VSMCs is an effective method of modulating the extent of intimal hyperplasia that occurs after balloon-induced vascular injury.

Adenoviridae↗
PubMed1997

p21CIP1-mediated inhibition of cell proliferation by overexpression of the gax homeodomain gene.

gax, a diverged homeobox gene expressed in vascular smooth muscle cells (VSMCs), is down-regulated in vitro by mitogen stimulation and in vivo in response to vascular injury that leads to cellular proliferation. Recombinant Gax protein microinjected into VSMCs and fibroblasts inhibited the mitogen-induced entry into S-phase when introduced either during quiescence or early stages of G1. Overexpression of gax with a replication-defective adenovirus vector resulted in G0/G1 cell cycle arrest of VSMCs and fibroblasts. The gax-induced growth inhibition correlated with a p53-independent up-regulation of the cyclin-dependent kinase inhibitor p21. Gax overexpression also led to an association of p21 with cdk2 complexes and a decrease in cdk2 activity. Fibroblasts deficient in p21 were not susceptible to a reduction in cdk2 activity or growth inhibition by gax overexpression. Localized delivery of the virus to denuded rat carotid arteries significantly reduced neointima formation and luminal narrowing. These data indicate that gax overexpression can inhibit cell proliferation in a p21-dependent manner and can modulate injury-induced changes in vessel wall morphology that result from excessive cellular proliferation.

Adenoviridae↗
PubMed1997

Evidence for the rapid onset of apoptosis in medial smooth muscle cells after balloon injury.

BACKGROUND: Vascular myocyte apoptotic cell death has been reported in human atherectomy and endarterectomy specimens and for neointimal smooth muscle cells (SMCs) in balloon-injured rat carotid arteries between 7 and 30 days after injury. However, the immediate effect of balloon injury on medial SMC viability has not been examined. METHODS AND RESULTS: Rat carotid arteries were harvested at the time of balloon injury (T = 0) and at 0.5, 1, 2, and 4 hours after injury. Uninjured vessels or vessels harvested at the time of injury (T = 0) did not display evidence of apoptosis. However, as early as 30 minutes after injury, 70% of medial SMCs appeared apoptotic by TdT-mediated dUTP nick end labeling (TUNEL) analysis and by the appearance of condensed chromatin. High frequencies of TUNEL-positive cells were also observed at 1 and 2 hours after injury but not at 4 hours. Transmission electron microscopy revealed many cells with morphological characteristics of apoptosis in the injured sections. A marked decrease in bcl-X expression was detected in the most luminal layers of the media. To corroborate these findings in a second animal model, rabbit external iliac arteries were analyzed after balloon angioplasty. Apoptotic cell death was evident in rabbit arteries at 30 minutes and at 4 hours after injury. CONCLUSIONS: As early as 30 minutes after balloon injury, myocytes appear to undergo apoptotic cell death at a high frequency as shown by TUNEL staining, chromatin condensation, and the appearance of morphological features in electron micrographs. The induction of apoptosis coincides with a marked downregulation of bcl-X expression.

Angioplasty, Balloon↗
PubMed1997

Rb functions to inhibit apoptosis during myocyte differentiation.

During in vitro myogenesis, a portion of myoblasts undergo apoptosis, whereas others continue with their differentiation program and form myotubes that are resistant to cell death. Previous work has shown that the expression of the Cdk inhibitor p21 correlates with enhanced resistance to apoptosis and that forced expression of p21 will confer this phenotype on differentiating myocytes. Here we examine the role of the retinoblastoma gene (Rb) in myocyte survival. Compared with wild-type myocytes, CC42 (Rb-/-) myocytes undergo higher frequencies of apoptosis during mitogen deprivation-induced myogenesis. Despite these features, Rb-/- myocytes display normal up-regulation of p21 and down-regulation of Cdk activities upon differentiation. Adenoviral constructs expressing the Cdk inhibitors p21 or p16 inhibit apoptosis in wild-type but not Rb-/- myocyte cultures. On the other hand, a Rb-expressing adenoviral construct inhibited apoptosis in both cell types. These data demonstrate that Rb functions downstream from the Cdk inhibitors to coordinate cell cycle withdrawal with programmed cell death during myocyte differentiation.

Animals↗
PubMed1997

Inhibition of myogenesis by multiple cyclin-Cdk complexes. Coordinate regulation of myogenesis and cell cycle activity at the level of E2F.

During skeletal myogenesis, cell cycle withdrawal accompanies the expression of the contractile phenotype. Here we show that ectopic expression of each D-type cyclin is sufficient to inhibit the transcriptional activation of the muscle-specific creatine kinase (MCK) gene. In contrast, ectopic expression of cyclin A or cyclin E inhibits MCK expression only when they are co-expressed with their catalytic partner cyclin-dependent kinase 2 (Cdk2). For each of these conditions, myogenic transcriptional inhibition is reversed by the ectopic co-expression of the general Cdk inhibitor p21. Inhibition of MCK expression by cyclins or cyclin-Cdk combinations correlates with E2F activation, suggesting that the inhibition is mediated by the overall Rb-kinase activities of the Cdk complexes. In support of this hypothesis, a hyperactive mutant of Rb was found to partially reverse the inhibition of MCK expression by cyclin D1 and by the combination of cyclin A and Cdk2. These data demonstrate that the inhibition of myogenic transcriptional activity is a general feature of overall Cdk activity which is mediated, at least in part, by an pocket protein/E2F-dependent pathway. MCK promoter activity is also inhibited by ectopic E2F1 expression, but this inhibition is not reversed by the co-expression of p21. Analyses of a series of E2F1 mutants revealed that the transcriptional activation, leucine zipper, basic, and cyclin A/Cdk2-binding domains are dispensable, but the helix-loop-helix region is essential for myogenic inhibition. These data demonstrate that myocyte proliferation and differentiation are coordinated at the level of E2F and that these opposing activities are regulated by different E2F domains.

Animals↗
PubMed1997

Prospects for intravascular gene therapy.

The goal of this review is to provide an overview of gene delivery systems and candidate genes currently being evaluated for genetic strategies in vascular gene therapy. We will discuss treatment strategies that have been shown by in vivo model systems to be efficacious in promoting neovascularization of ischemic tissue or limiting post-interventional restenosis by inhibiting smooth muscle cell proliferation and/or encouraging re-endothelialization.

Animals↗
PubMed1997

Coordinate regulation of cell cycle and apoptosis during myogenesis.

During myogenesis, precursor cells irreversibly withdraw from the cell cycle as they differentiate into mature myotubes. The state of myocyte differentiation also influences the propensity of these cells to undergo apoptosis. Proliferative precursor cells are far more susceptible to apoptotic cell death than are terminally differentiated myotubes. The upregulation of the cdk inhibitor p21 and the dephosphorylation of pRb are critical regulatory events that establish both the post-mitotic and apoptosis-resistant states. The coordinate regulation of cell proliferation and death provides the organism with a mechanism to control the deposition of muscle mass during embryonic development.

Animals↗
PubMed1997

Percutaneous delivery of the gax gene inhibits vessel stenosis in a rabbit model of balloon angioplasty.

OBJECTIVES: The expression of gax, an anti-proliferative homeobox gene, is rapidly downregulated in vascular smooth muscle cells (VSMCs) following arterial injury. Here we performed percutaneous adenovirus-mediated gene transfer into the iliac arteries of normal rabbits using a channel balloon catheter to assess the effects of gax overexpression on neointima formation, lumen diameter, reendothelialization and functional vasomotion. METHODS: A channel balloon catheter was used to perform both the arterial injury and local gene delivery. In each animal both iliac arteries were randomly assigned to receive either an adenovirus expressing the gax gene (Ad-Gax) or the beta-galactosidase gene (Ad-beta gal). In a second group of animals arteries were randomly assigned to receive either Ad-beta gal or saline. RESULTS: At one month, angiography revealed 36% less luminal narrowing in the Ad-Gax-treated arteries relative to the Ad-beta gal-treated control arteries. Histological analysis revealed that the intimal/medial ratio (I/M) was reduced by 56% in the Ad-Gax group. Endothelium-dependent vasomotion was not affected by the gax gene transfer. In the second group, no statistically significant differences were found between the saline and the Ad-beta gal-treated vessels for any of these parameters. CONCLUSIONS: Percutaneous adenovirus delivery of the gax gene to rabbit iliac arteries following endothelial denudation and vessel wall injury reduces neointimal hyperplasia and luminal stenosis, but does not affect endothelium-dependent vasomotion. This study demonstrates that a VSMC transcription factor can potentially be utilized for the development of a molecular therapy for vascular disorders.

Adenoviridae↗
PubMed1997

Cell cycle exit upon myogenic differentiation.

Myogenic precursor cells withdraw irreversibly from the cell cycle as they differentiate into mature myotubes. Cell cycle exit occurs early during the differentiation program and is required for normal expression of the contractile phenotype. Differentiated myocytes also display a decreased propensity to undergo apoptotic cell death. The upregulation of the cyclin-dependent kinase inhibitor p21 and the dephosphorylation of retinoblastoma protein (pRb) appear to be critical regulatory events for the establishment of both the postmitotic and apoptosis-resistant states. The coordinated regulation of cell proliferation and death provides the developing embryo with a mechanism for controlling muscle mass and thereby the size of individual motor units.

Animals↗
PubMed1997

Relationship between volumes and areas from single transverse scans of intra-abdominal fat measured by magnetic resonance imaging.

OBJECTIVE: To determine the level of a single transverse scan of intra-abdominal fat between L1 and L5 vertebrae that best predicts intra-abdominal fat volumes. SUBJECTS: Sixteen male and seven female patients with non-insulin-dependent diabetes mellitus, aged 44-74 y. OUTCOME MEASURES: Volumes and areas from single scans of intra-abdominal fat measured by magnetic resonance imaging with a 1.5 Tesla magnetic field strength. RESULTS: Intra-abdominal fat volumes and were calculated from fat areas from eight cross-sectional transverse single scans (nine scans in eight men) of 20 mm thickness. Men and women, respectively, had mean body mass index (BMI) of 27.9 (s.d. 3.0) and 31.6 (s.d. 4.7) kg/m2, and intra-abdominal fat of 2.3 (s.d. 0.5) and 2.5 (s.d. 0.6) kg. Intra-abdominal fat area of the fourth scan (in the direction of L1 to L5) gave the highest prediction of total intra-abdominal fat both in men (r = 0.959, P < 0.001) and in women (r = 0.973, P < 0.001). The intra-abdominal fat area of the third scan gave almost as good a prediction. These third and fourth scans corresponded to L2 and L3 vertebrae. The intra-abdominal fat areas from the sixth and seventh scans, corresponded to the frequently used L4-L5 and had lower correlations with intra-abdominal fat. There were no gender differences in the prediction of volumes from areas of intra-abdominal fat. Intra-abdominal fat areas of the fourth scan explained 93% of variance (SEE = 0.14 kg) of total of intra-abdominal fat for both genders: intra-abdominal fat (kg) = 0.0108 x intra-abdominal fat area of the fourth scan (cm2) + 0.244. CONCLUSIONS: In large studies of intra-abdominal fat, using magnetic resonance imaging (MRI) or computerised tomography scanning, a single intra-abdominal fat area at the intervertebral disc between L-2 and L-3 vertebrae offers a cheaper, faster and safer method, with high prediction of total intra-abdominal fat volumes and masses.

Abdomen↗
PubMed1997

Encounters, endings and temporality in psychiatric nursing.

This paper is derived from data obtained from a study in progress. The study is a phenomenological research project grounded in the work of Martin Heidegger and Hans-George Gadamer. Psychiatric nurses were interviewed about their experience of the nurse-patient encounter. During the course of the study it became apparent that the significance of encounters did not 'end' with the termination of the relationship. 'Endings' went further than notions of relationship 'termination' familiar in the psychiatric nursing literature. The ending of the encounter seemed to encapsulate much of the significance of the encounter for the nurse. It also became apparent that, for these nurses, time was not experienced in a linear way. Viewed in this non-linear way, the past is no less meaningful for being past. The past is with us and influences the present, that is, our actions now. The future is no less meaningful for having not yet been. The future engages us in possibilities. So, unlike the narrow concept of termination, the lived experience of endings captures the totality of the relationship and the encounter and therefore lives on in the present. In the lived experience of endings the past and the future are both significant and have meaning.

Anger↗
PubMed1997

A competitive mechanism of CArG element regulation by YY1 and SRF: implications for assessment of Phox1/MHox transcription factor interactions at CArG elements.

In the promoters of many immediate early genes, including c-fos, CArG DNA regulatory elements mediate basal constituitive expression and rapid and transient serum induction. CArG boxes also occur in the promoters of muscle-specific genes, including skeletal alpha-actin, where it confers muscle-specific expression. These elements are regulated, at least in part, by the ubiquitous transcription factors serum response factor (SRF) and YY1. The homeobox transcription factor Phox1/MHox has also been implicated in regulation of the c-fos CArG element and is thought to function by facilitating SRF binding to DNA. Here, we provide in vitro and in vivo evidence that the mechanism of YY1 repression of CArG elements results from competition with SRF for overlapping binding sites. We describe in detail the binding sites of YY1 and SRF through serial point mutations of the skeletal alpha-actin proximal CArG element and identify a mutation that dramatically reduces YY1 binding but retains normal SRF binding. YY1 competes with SRF for binding to wild-type CArG elements, but not to this point mutant in vitro. This mutant is sufficient for muscle-specific expression in vivo but is much less sensitive to repression by YY1 overexpression. We utilized the YY1/SRF competition to address the role of Phox1 at these elements. Phox1 overexpression did not diminish YY1-mediated repression, suggesting that transcriptional activation by Phox1 does not result from enhanced SRF binding to these elements. These methods may prove to be useful for assessing interactions between other CArG element regulatory factors.

Actins↗
PubMed1997

Dissociative symptoms in panic disorder.

The present study examined the prevalence and correlates of dissociative symptoms in patients with panic disorder and patients with other nonpanic anxiety disorders. A total of 56 patients with anxiety disorders (13 with panic disorder alone, 16 with comorbid panic and other anxiety, and 27 with other anxiety disorders) were assessed with structured clinical interviews and a battery of questionnaires. Although 69% of patients with panic disorder experienced depersonalization or derealization during their panic attacks, panic disorder patients were no more likely to experience dissociative experiences as assessed by the Dissociative Experience Scale than patients with other anxiety disorders. In the entire sample, the prevalence of dissociative experiences was very low and well within nonpathological ranges. The correlates of dissociative symptoms were severity of depression, social anxiety, and personality disorders. The implications of these findings for conceptualizing the nature of dissociative symptoms within an anxiety population are discussed.

Adult↗
PubMed1997

Changing handover practices: one private hospital's experiences.

The handover practice has long been an important component of clinical nursing practice allowing nurses to exchange relevant client information from one shift to the next and ensure continuity of patient care. Traditional approaches have seen nursing handovers taking place in a room away from general ward activity. Oncoming nursing staff receive the information verbally from nurses on the previous shift about all patients within the ward or unit. This practice has been proven over time to present difficulties and consequently, many hospitals are choosing to adopt models that better address current needs. This analysis describes the creative approaches taken by one private hospital in modifying handover practices with the view to reduce time and increase overall efficiency and effectiveness, whilst ensuring that staff and ward requirements are considered. The study highlights how action research principles can be applied to introduce change into the clinical practice environment.

Communication↗
PubMed1997

Embryonic expression of the Gax homeodomain protein in cardiac, smooth, and skeletal muscle.

Gax is a homeobox-containing gene that has been detected in adult cardiovascular tissues and exhibits a growth arrest-specific pattern of expression in cultured vascular myocytes. To study the regulation of gax during development, we performed immunohistochemistry and in situ hybridization on mouse embryos. Gax was present in mesodermally and, as with other homeobox genes, neuroectodermally derived tissues. Early mesodermal protein expression was limited to the lateral plate and somitic mesoderm. Gax in the cardiac muscle lineage exhibited a biphasic pattern of expression. Expression was prominent in the heart tube of the earliest cardiomyocytes and remained prominent through the looping stage (day 12.5 post coitum [pc]) but fell below the threshold of detection in atria and ventricles by day 13.5 pc. At day 15.5 pc, Gax protein was again detectable but restricted to cells within the compact layer of the ventricular myocardium. Gax expression was also noted in smooth muscle cells as early as day 9.5 pc. In the skeletal muscle lineage, Gax protein was expressed at the onset of somitogenesis before the expression of the myogenic basic helix-loop-helix and MEF2/RSRF family proteins. Subsequently, it was noted at day 9.5 pc in premyogenic cells migrating into head, trunk, and limb buds. Gax was detected in myotomes, premuscle masses, and mature muscle groups. These data suggest an important developmental role for Gax in all muscle lineages.

Animals↗
PubMed1997

Forced expression of the homeodomain protein Gax inhibits cardiomyocyte proliferation and perturbs heart morphogenesis.

The development of the tubular heart into a complex four-chambered organ requires precise temporal and region-specific regulation of cell proliferation, migration, death and differentiation. While the regulatory mechanisms in heart morphogenesis are not well understood, increasing attention has focused on the homeodomain proteins, which are generally linked to morphogenetic processes. The homeodomain containing gene Gax has been shown to be expressed in heart and smooth muscle tissues. In this study, the Gax protein was detected in the nuclei of myocardial cells relatively late in chicken heart development, at a time when myocyte proliferation is declining. To test the hypothesis that the Gax protein functions as a negative regulator of cardiomyocyte proliferation, a replication-defective adenovirus was used to force its precocious nuclear expression during chicken heart morphogenesis. In experiments in which Gax- and beta-galactosidase-expressing adenoviruses were co-injected, clonal expansion of myocytes was reduced, consistent with inhibition of myocyte proliferation. This effect on proliferation was corroborated by the finding that the percentage of exogenous Gax-expressing myocytes that were positive for the cell cycle marker PCNA decreased over time and was lower than in control myocytes. The precocious nuclear expression of Gax in tubular hearts resulted in abnormal heart morphology, including small ventricles with rounded apices, a thinned compact zone and coarse trabeculae. These results suggest a role for the Gax protein in heart morphogenesis causing proliferating cardiomyocytes to withdraw from the cell cycle, thus influencing the size and shape that the heart ultimately attains.

Adenoviridae↗
PubMed1997

Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty.

Intimal hyperplasia following angioplasty results in part from the migration and proliferation of vascular smooth muscle cells (VSMCs). However, the cell cycle regulatory networks underlying injury-induced VSMC proliferation are largely unknown. In the present study, we examined the kinetics of expression and activity of cell cycle regulatory factors after angioplasty in rat and human arteries. Cell lysates were prepared from uninjured rat carotid arteries and at different time points after balloon denudation. Marked induction of the proliferating cell nuclear antigen (PCNA), the G1/S cyclin-dependent kinase (cdk2), and its regulatory subunits (cyclin E and cyclin A) occurred between 1 and 2 days after angioplasty, was sustained up to 10 days after injury, and then declined. Induction of these factors correlated with increased cdk2-, cyclin E-, and cyclin A-dependent kinase activity, indicating the assembly of functional cdk2/cyclin E and cdk2/cyclin A holoenzymes in the injured arterial wall. Immunohistochemical analysis revealed early expression of cdk2, cyclin E, and PCNA within the media of injured carotid arteries. At later time points, expression of these markers declined to basal levels in the media but was detected within the intimal lesion. Thus, VSMC proliferation after angioplasty in the rat carotid artery is associated with a temporally and spatially coordinated expression of cdk2, cyclins E and A, and PCNA. Analysis of human arteries also revealed expression of these factors in VSMCs within restenotic lesions. Thus, cdk2 and its regulatory cyclins may be suitable targets to limit human restenosis.

Actins↗