Back pain in Britain: comparison of two prevalence surveys at an interval of 10 years.
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Adult↗
Biomedical subjects
K Walsh
Publications and source records attributed to K Walsh.
Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt.
Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding beta-galactosidase (beta-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with beta-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of beta-gal-infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.
Actins↗
Signal transducer and activator of transcription 3 is required for glycoprotein 130-mediated induction of vascular endothelial growth factor in cardiac myocytes.
Activation of glycoprotein (gp) 130 transduces hypertrophic and cytoprotective signals in cardiac myocytes. In the present study, we have demonstrated that signals through gp130 increase the expression of vascular endothelial growth factor (VEGF) in cardiac myocytes via the signal transducer and activator of transcription (STAT) 3 pathway. After activation of gp130 with leukemia inhibitory factor (LIF), expression of VEGF mRNA rapidly increased with a peak at 3 h in cultured cardiac myocytes. Cardiotrophin-1 also enhanced VEGF mRNA expression in a dose-dependent manner. VEGF protein production and secretion to the medium were also enhanced by LIF and cardiotrophin-1 but not by interleukin-6. Adenovirus transfer of the dominant-negative form of STAT3 to cultured cardiac myocytes inhibited induction of VEGF expression induced by LIF, but neither PD98059 nor wortmannin was affected. In murine hearts, intravenous administration of LIF augmented expression of VEGF mRNA; however, the hearts of transgenic mice overexpressing dominant-negative STAT3 showed reduced expression of VEGF mRNA that was not induced after LIF stimulation. These data provide the first evidence that a STAT family protein functions as a regulator of angiogenic growth factors and suggest that gp130/STAT signaling in cardiac myocytes can control vessel growth during cardiac remodeling.
Animals↗
Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart.
BACKGROUND: IGF-1 has been shown to protect myocardium against death in animal models of infarct and ischemia-reperfusion injury. In the present study, we investigated the role of the IGF-1-regulated protein kinase Akt in cardiac myocyte survival in vitro and in vivo. METHODS AND RESULTS: IGF-1 promoted survival of cultured cardiomyocytes under conditions of serum deprivation in a dose-dependent manner but had no effect on cardiac fibroblast survival. The cytoprotective effect of IGF-1 on cardiomyocytes was abrogated by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Wortmannin had no effect on cardiomyocyte viability in the absence of IGF-1. IGF-1-mediated cytoprotection correlated with the wortmannin-sensitive induction of Akt protein kinase activity. To examine the functional consequences of Akt activation in cardiomyocyte survival, replication-defective adenoviral constructs expressing wild-type, dominant-negative, and constitutively active Akt genes were constructed. Transduction of dominant-negative Akt blocked IGF-1-induced survival but had no effect on cardiomyocyte survival in the absence of IGF-1. In contrast, transduction of wild-type Akt enhanced cardiomyocyte survival at subsaturating levels of IGF-1, whereas constitutively active Akt protected cardiomyocytes from apoptosis in the absence of IGF-1. After transduction into the mouse heart in vivo, constitutively active Akt protected against myocyte apoptosis in response to ischemia-reperfusion injury. CONCLUSIONS: These data are the first documentation that Akt functions to promote cellular survival in vivo, and they indicate that the activation of this pathway may be useful in promoting myocyte survival in the diseased heart.
Animals↗
Analysis of adenoviral transport mechanisms in the vessel wall and optimization of gene transfer using local delivery catheters.
Local delivery devices have been used for adenovirus-mediated gene transfer to the arterial wall for the potential treatment of vascular proliferative diseases. However, low levels of adenoviral gene expression in vascular smooth muscle cells may pose a serious limitation to the success of these procedures in the clinic. In this study, we examined the mechanisms controlling adenoviral transport to the vessel wall, using both hydrogel-coated and infusion-based local delivery catheters, with the goal of enhancing in vivo gene transfer under clinically relevant delivery conditions. The following delivery parameters were tested in vivo: applied transmural pressure, viral solution volume and concentration, and delivery time. We found that viral particles are transported into the vessel wall in a manner consistent with diffusion rather than pressure-driven convection. Consistent with diffusion, viral concentration was shown to be the key variable for viral transport in the vessel wall and thus gene expression in vascular smooth muscle cells. A transduction level of 17.8+/-3.2% was achieved by delivering a low volume of concentrated adenoviral beta-galactosidase solution through an infusion balloon catheter at low pressure without an adverse effect on medial cellularity. Under these conditions, effective gene transfer was accomplished within a clinically relevant time frame of 2 min, indicating that longer delivery times may not be necessary to achieve efficient gene transfer.
Adenoviridae↗
Fas ligand-deficient mice display enhanced leukocyte infiltration and intima hyperplasia in flow-restricted vessels.
Fas ligand (FasL) is a death factor that induces apoptosis in Fas-bearing cells. To explore the role of FasL in vascular lesion formation, we analysed leukocyte infiltration and lesion formation in a flow-restriction model of vascular injury that results in neointima formation in the presence of intact endothelium. The left common carotid arteries of wild-type and FasL-deficient (gld) mice were ligated just proximal to the carotid bifurcation. Three days after the ligation, T lymphocyte and macrophage infiltration into the common carotid artery was notably enhanced in the gld mice relative to the wild-type C57BL/6J mice. Four weeks after the ligation, the common carotid arteries developed neointima-like lesions consisting primarily of alpha -smooth muscle actin-positive cells beneath an endothelial cell monolayer. Neointima formation was greater in the gld mice than in wild-type mice. These data provide mouse genetic evidence suggesting that Fas-mediated cell death can function to restrict inflammation and intimal hyperplasia during vascular remodelling.
Actins↗
Building a better mouse model.
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Animals↗
Intracoronary, adenovirus-mediated Akt gene transfer in heart limits infarct size following ischemia-reperfusion injury in vivo.
BACKGROUND: Previous data have shown that enhanced Akt signaling inhibits cardiac myocyte apoptosis in vitro and in vivo. To elucidate the contribution of apoptosis to the pathogenesis of the infarct, we investigated whether intra-coronary Akt gene delivery could reduce gross infarct size following ischemia/reperfusion injury. METHODS AND RESULTS: Replication-defective adenoviral constructs encoding a myristoylated, constitutively-active form of Akt (myrAkt) or beta -galactosidase were delivered to rat hearts by intracoronary perfusion. Twenty-four h after gene transduction, hearts in both groups underwent 45 min of ischemia followed by 4 h of reperfusion. A third group of animals also underwent ischemia-reperfusion injury but were not transduced with an adenoviral vector. The proportion of the left ventricle at risk was not different among the experimental groups. However, infarct size as a proportion of the area at risk was significantly lower in myrAkt-treated group than in the beta -galactosidase treated group or in the control group that was not subject to intracoronary perfusion (myrAkt=20.9+/-2.7%v beta -galactosidase=56.1+/-3.9% and control=46.2+/-4.6%, P<0.05), as was infarct size as a proportion of the total left ventricle (myrAkt=11.4+/-3.2 v beta -galactosidase=32. 9+/-3.3 and control=23.5+/-3.0, P<0.05). CONCLUSIONS: These data demonstrate that Akt signaling limits infarct size following ischemia/reperfusion injury and they indicate that the activation of this pathway may be useful in protecting against myocardial loss in the diseased heart.
Adenoviridae↗
Gene therapy for restenosis.
This review provides an overview of candidate genes that are currently being evaluated for genetic strategies in vascular gene therapy. We discuss treatment strategies that have proven efficacious in limiting postinterventional restenosis through evaluation with in vivo model systems. The candidate strategies utilize genes that are either cytotoxic, regulate vascular smooth muscle cell differentiation or proliferation. In addition, we review oligonuclotide and ribozyme strategies that function by suppressing the expression of cell cycle regulators.
Animals↗
Apoptosis in inflammatory-fibroproliferative disorders of the vessel wall.
Apoptotic cell death is a hallmark of inflammatory-fibroproliferative disorders of the vessel wall. Here, we review what is currently known about cell death within atherosclerotic and restenotic lesions. We also examine evidence suggesting that inflammatory cells contribute to the regulation of cell turnover within these lesions, and discuss the molecules expressed by vascular cells that modulate these processes. In toto, these studies suggest that apoptosis is prevalent in vascular lesions, controlling the viability of both inflammatory and vascular cells, and thus determining the cellular composition of the vessel wall.
Apoptosis↗
Adenovirus-encoded hammerhead ribozyme to Bcl-2 inhibits neointimal hyperplasia and induces vascular smooth muscle cell apoptosis.
OBJECTIVE: The balance between apoptosis and cell proliferation is vital for cellular homeostasis, yet little is known about the mechanisms that coordinate these two cell fates, particularly in the vessel wall. It is well established that the members of Bcl-2-gene family are regulators of apoptosis, but their role in cellular proliferation is less clear. METHODS: We analyzed the effects of disrupting Bcl-2 expression in vascular smooth muscle cells (VSMCs) by adenoviral-mediated delivery of a hammerhead ribozyme against bcl-2 mRNA (Ad-Rbz-Bcl-2). RESULTS: Forced ablation of Bcl-2 in balloon-injured rat carotid arteries reduced cell number and inhibited neointimal hyperplasia. In vitro, VSMCs transduced with the Ad-Rbz-Bcl-2 underwent apoptosis as indicated by a reduction in cell number and DNA fragmentation. Ad-Rbz-Bcl-2-transduced cells also exhibited aberrations in both G1- and S-phases of the cell cycle. However, forced perturbations in cell cycle activity by serum-stimulation or treatment with chemical inhibitors did not affect Ad-Rbz-Bcl-2-induced cell death, indicating that these cell cycle changes are not essential for apoptosis. CONCLUSION: These data show that physiological levels of Bcl-2 are essential for VSMC viability and that ablation of Bcl-2 alters cell cycle activity through the execution of the apoptotic process.
Adenoviridae↗
The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals.
Recent studies suggest that statins can function to protect the vasculature in a manner that is independent of their lipid-lowering activity. We show here that statins rapidly activate the protein kinase Akt/PKB in endothelial cells. Accordingly, simvastatin enhanced phosphorylation of the endogenous Akt substrate endothelial nitric oxide synthase (eNOS), inhibited apoptosis and accelerated vascular structure formation in vitro in an Akt-dependent manner. Similar to vascular endothelial growth factor (VEGF) treatment, both simvastatin administration and enhanced Akt signaling in the endothelium promoted angiogenesis in ischemic limbs of normocholesterolemic rabbits. Therefore, activation of Akt represents a mechanism that can account for some of the beneficial side effects of statins, including the promotion of new blood vessel growth.
Animals↗
Effect of percutaneous adenovirus-mediated Gax gene delivery to the arterial wall in double-injured atheromatous stented rabbit iliac arteries.
Though the efficacy of intravascular gene transfer has been demonstrated in native vessels following acute injury, this methodology has not been validated in complex models of vascular injury that more closely mimic clinical angioplasty procedures. Previous studies have shown that Gax gene overexpression modulates the injury-induced remodeling of the vessel in rat carotid and normal rabbit iliac arteries. Here, we evaluated the effect of the Gax gene delivery in atheromatous stented vessels. Rabbits were fed 120 g daily of 1% cholesterol diet for 3 weeks. At 1 week they underwent initial injury on the external iliac artery, then balloon angioplasty was performed at 3 weeks at the same site with a 2.5 mm diameter channel balloon catheter (three times 1 min at 6 atm). Either saline (n = 4) or the control viral construct Ad-CMVluc (5 x 109 p.f.u.) (n = 5) or Ad-CMVGax (5 x 10(9) p.f.u.) (n = 4) was delivered with a poloxamer mixture via a channel balloon (6 atm, 30 min), and a 15 mm long Palmaz-Schatz stent (PS154) was then deployed at the site (1 min, 8 atm). Arteries were analyzed 1 month later. At 1 month, the Ad-CMVGax treated arteries exhibited a lower maximal intimal area (1. 15+/-0.1 mm2) than saline (1.87+/-0.15 mm2, P = 0.007) or Ad-CMVluc-treated vessels (1.98+/-0.31 mm2, P = 0.04). Likewise Ad-CMVGax-treated vessels displayed a lower maximal percentage cross-sectional area narrowing (35.1+/-3.5%) than saline (65.3+/-9.4%, P = 0.01) or Ad-CMVluc-treated vessels (62.7+/-6.7%, P = 0.02). Angiographic analysis revealed larger minimal lumen diameter in Ad-CMVGax treated arteries (2.0+/-0.1 mm) than saline (1.14+/-0.36 mm, P = 0.06) or Ad-CMVluc-treated vessels (1.23+/-0.25 mm, P = 0.02). Overexpression of the Gax gene inhibits neointimal hyperplasia and lumen loss in atheromatous stented rabbit iliac arteries.
Adenoviridae↗
The detection of prostate cells by the reverse transcription-polymerase chain reaction in the circulation of patients undergoing transurethral resection of the prostate.
OBJECTIVE: To determine whether prostate cells are disseminated into the circulation of patients after transurethral resection of the prostate (TURP), as assessed by a reverse transcription-polymerase chain reaction (RT-PCR) assay for prostate specific antigen (PSA) mRNA. PATIENTS AND METHODS: Fifty-one patients, comprising 34 with benign prostatic hyperplasia (BPH) and 17 with prostate cancer who were undergoing routine TURP, had blood samples taken before and 30 min after surgery. The blood mononuclear cell layer was isolated by density-gradient centrifugation and total RNA extracted. Complementary DNA was synthesized by RT of the RNA. The target PSA sequence was amplified by PCR with specific PSA primers and the product detected on agarose-gel electrophoresis with ethidium bromide staining. RESULTS: Five patients (all with prostate cancer) were positive on PSA RT-PCR before surgery and remained positive after TURP. Of the remaining 46 patients, five (11%; three with BPH and two with cancer) changed from a negative to a positive result after TURP. CONCLUSION: In a proportion of patients TURP causes the dissemination of prostate cells into the circulation which are then detectable by RT-PCR of PSA mRNA.
Aged↗
Troglitazone halts diabetic glomerulosclerosis by blockade of mesangial expansion.
BACKGROUND: Renal complications of long-term, poorly controlled type 2 diabetes mellitus include glomerulosclerosis and interstitial fibrosis. The onset and progression of these complications are influenced by underlying pathophysiologies such as hyperglycemia, hypertriglyceridemia, and hypercholesterolemia. Troglitazone, a thiazolidinedione, has been shown to ameliorate these metabolic defects. However, it was not known whether therapeutic intervention with troglitazone would prevent the onset and progression of glomerulosclerosis. METHODS: Sixty male ZDF/Gmitrade mark rats and 30 age-matched Zucker lean rats were in the study. The ZDF/Gmitrade mark rats were divided into two groups, one in which blood glucose levels were uncontrolled (30 animals) and another (30) in which blood glucose was controlled via dietary administration of troglitazone. Ten animals from each group were sacrificed at one, three, and six months into the study. The kidneys were harvested and processed for immunostaining with BM-CSPG, a marker for mesangial matrix. Images of 200 glomeruli per animal were captured using digital imaging microscopy, and the index of mesangial expansion (total area mesangium/total area of tuft) per glomerular section was measured. RESULTS: The administration of troglitazone ameliorated the metabolic defects associated with type 2 diabetes mellitus. Moreover, the glomeruli from tissue sections of animals given troglitazone showed no mesangial expansion when compared with normoglycemic control animals, whereas the uncontrolled diabetic animals showed significant mesangial expansion at all time intervals. CONCLUSIONS: Therapeutic intervention with the thiazolidinedione troglitazone halts the early onset and progression of mesangial expansion in the ZDF/Gmitrade mark rat, preventing the development of glomerulosclerosis in this animal model of type 2 diabetes mellitus.
Animals↗
Alcoholic liver disease.
Alcohol is a major cause of liver cirrhosis in the Western world and accounts for the majority of cases of liver cirrhosis seen in district general hospitals in the UK. The three most widely recognised forms of alcoholic liver disease are alcoholic fatty liver (steatosis), acute alcoholic hepatitis, and alcoholic cirrhosis. The exact pathogenesis of alcoholic liver injury is still not clear but immune mediated and free radical hepatic injury are thought to be important. There is increasing interest in genetic factors predisposing to hepatic injury in susceptible individuals. Diagnosis is based on accurate history, raised serum markers such as gamma-glutamyltransferase, mean corpuscular volume, and IgA and liver histology when obtainable. Abstinence is the most important aspect of treatment. Newer drugs such as acamprosate and naltrexone are used to reduce alcohol craving. Vitamin supplements and nutrition are vital while corticosteroids have a role in acute alcoholic hepatitis where there is no evidence of gastrointestinal haemorrhage or sepsis. Liver transplantation has excellent results in abstinent patients with end stage liver disease but there are concerns about recidivism after transplant.
Cytokines↗
Vascular endothelial cells and smooth muscle cells differ in expression of Fas and Fas ligand and in sensitivity to Fas ligand-induced cell death: implications for vascular disease and therapy.
Fas ligand (FasL) is a death factor that induces apoptosis in cells bearing its receptor, Fas. Fas and FasL have been detected in the vessel wall, and it has been proposed that Fas-mediated apoptosis has a role in physiological and pathological cell turnover in the vasculature. Here, we evaluated the expression of Fas in the presence and absence of cytokines on both endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). We also examined the sensitivity of ECs and VSMCs to Fas-mediated apoptosis induced by exposure to multiple Fas agonists: soluble FasL, anti-Fas antibody, and membrane-bound FasL resulting from transduction with a replication-defective adenovirus expressing FasL (Adeno-FasL). Cell-surface FasL expression was detected on human ECs with the use of 4 anti-FasL antibodies, whereas cell-surface FasL expression was not detected on VSMCs. Unstimulated ECs expressed relatively low levels of Fas, but expression was upregulated after treatment with tumor necrosis factor-alpha (TNF-alpha) or interferon gamma (IFN-gamma). In contrast, VSMCs expressed relatively high levels of Fas, and treatment with TNF-alpha or IFN-gamma induced little or no upregulation under the conditions of these assays. ECs were resistant to death after exposure to soluble FasL or agonist anti-Fas antibody and also after infection with Adeno-FasL in the presence or absence of cytokine treatment. In contrast, VSMCs remained viable in the presence of soluble FasL or agonist anti-Fas antibody, but they underwent apoptosis after infection with Adeno-FasL. IFN-gamma enhanced Adeno-FasL-induced death of VSMCs, but TNF-alpha did not. These findings provide insights about the potential role of Fas-mediated apoptosis in the vessel wall and suggest strategies to treat proliferative vascular diseases by exploiting the differential sensitivity of ECs and VSMCs to FasL-induced cell death.
Apoptosis↗
Regulation of cdk2 activity in endothelial cells that are inhibited from growth by cell contact.
Endothelial cells (ECs) are quiescent in normal blood vessels but undergo rapid bursts of proliferation after vascular injury and during angiogenesis. Here we show that the activity of cyclin-dependent kinase-2 (cdk2), a key regulator of the G1 and S phases of the cell cycle, is expressed at high levels in proliferating ECs but at low levels in ECs that are contact-inhibited for growth. Despite these differences in kinase activity, the protein levels of cdk2 and 1 of its activating subunits, cyclin E, are not modulated by these different growth conditions. The cdk inhibitor p27 is highly expressed in contact-inhibited but not proliferating ECs, whereas the level of cyclin A protein is preferentially expressed in proliferating ECs. p27 protein was detected in immunoprecipitable complexes with cdk2 or cyclin E in cultures that were contact-inhibited for growth. The functional significance of the p27 induction was indicated by the detection of a heat-stable cdk2 inhibitory activity that was induced by endothelial cell-cell contact and could be immunodepleted with anti-p27 antibodies. In a confluent EC monolayer, cdk2 kinase activity was activated by a scraping injury that led to cell migration and proliferation. The injury-induced activation of cdk2 coincided with the downregulation of p27 and the induction of cyclin A. These data demonstrate that p27 is induced in confluent cultures of ECs. They also indicate that both p27 induction and cyclin A downregulation contribute to the inhibition of cdk2 and cell proliferation by cell-cell contact in ECs.
Animals↗