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Biomedical subjects

K Walsh

Publications and source records attributed to K Walsh.

At least 37 records · Page 2Linked to original sources
PubMed2002

Distribution of vascular endothelial growth factor (VEGF) in prostate disease.

Vascular endothelial growth factor (VEGF) is a heparin-binding polypeptide growth factor. It is a potent mitogen for endothelial cells. Immunohistochemical localisation of VEGF was performed on 25 moderate to poorly differentiated stage T4 M+ prostate cancer specimens and 30 benign prostatic hyperplasia (BPH) specimens. A positive result was indicated by area staining >25% and +2 or +3 staining intensity. Positive epithelial staining was observed in 50% of BPH specimens and 56% of cancer specimens, while positive stromal staining was observed in 73% of BPH specimens and 30% of cancer specimens. This may reflect an active role for stromal VEGF in the pathological process of BPH.

Endothelial Growth Factors↗
PubMed2002

Decorin affects endothelial cells by Akt-dependent and -independent pathways.

Decorin, a small multifunctional proteoglycan, has been shown to be causally involved in the formation of capillary-like structures and a decrease in apoptosis. Here we investigated signal transduction pathways mediating effects of decorin on endothelial cells (ECs). Addition of decorin led to a fourfold increase in phosphorylation of Akt/protein kinase B on Thr307 and a l.4-fold increase on Ser473 after 10 min, but this phosphorylation could not be blocked by preincubation with Ly29400 (10 micro M). Six hours after the addition of decorin, the synthesis of p21 and p27, two inhibitors of cyclin-dependent kinases, started and increased up to 18 h, while synthesis of cyclin A peaked at 12 h and decreased after 24 h below base level. Induction of dominan-negative Akt by a replication-deficient adenovirus blocked p21 and cyclin A synthesis, but had no effect on p27. Dominant-negative Akt also blocked the antiapoptotic effect of decorin on ECs, but induction of dominant-positive Akt could not rescue the cells from apoptosis. Thus, the matrix proteoglycan decorin is a signaling molecule in ECs that affects cell survival by Akt-dependent and -independent pathways.

Animals↗
PubMed2002

An algorithm for the investigation and management of patients with suspected deep venous thrombosis at a district general hospital.

The aim was to show that clinical assessment and serial Dopplers could be used to exclude deep venous thrombosis (DVT), and also that clinical assessment in combination with D-dimers could be used to exclude DVT. This was a prospective trial. Patients underwent clinical assessment, colour flow Doppler, and D-dimer measurement. Patients with an equivocal first Doppler and patients with a moderate/high pre-test clinical score had a repeat Doppler. Follow up information was obtained to see if further thromboembolism developed. Outcome measures included the reliability of the clinical assessment, Doppler, and D-dimer in excluding DVT. One hundred and ninety four patients were enrolled. The negative predictive value of a low medical pre-test score in excluding DVT was 92%. Eighty eight patients underwent D-dimer measurement. The negative predictive value of a D-dimer <400 units was 96%. The negative predictive value of a low clinical pre-test score and D-dimer <400 units was 100%. The negative predictive value of a low clinical pre-test score and D-dimer <800 units was 97%. The negative predictive value of the combination of a low clinical pre-test score and D-dimer <400 units was 100%. Twenty three per cent of patients had a low clinical pre-test score and D-dimer <400 units. Thus, a DVT could be excluded in 23% of referred patients. This information could be used to reduce Doppler requests by 23%. No patient in whom clinical assessment and serial Dopplers had excluded a DVT suffered further thromboembolism on follow up. This study has thus shown that a low medical pre-test score and negative Doppler reliably exclude DVT; a low pre-test score and negative D-dimer would reliably exclude DVT.

Adult↗
PubMed2001

Decorin-mediated signal transduction in endothelial cells. Involvement of Akt/protein kinase B in up-regulation of p21(WAF1/CIP1) but not p27(KIP1).

Endothelial cells undergoing angiogenesis express decorin, a small multifunctional proteoglycan. We have shown that decorin is causally involved in the formation of capillary-like structures and a decrease in apoptosis in endothelial cells cultured in a collagen lattice. Here we investigate signal transduction pathways mediating the effects of decorin. Reverse transcription-polymerase chain reaction demonstrated that p21 and p27, two inhibitors of cyclin-dependent kinases, were up-regulated by decorin induction. Decorin also increased protein levels of p21 and caused its translocation into the nucleus. p21 synthesis started 6 h after decorin addition and reached a plateau after 18 h, while cyclin A, which was also induced, peaked at 12 h and declined below basal levels within 24 h. These effects were mediated by the Akt/protein kinase B pathway. Akt phosphorylation at Thr-308 increased 4-fold and at Ser-473 1.4-fold within 10 min after decorin addition. Overexpression of dominant negative Akt inhibited the decorin-mediated induction of p21 and cyclin A, but had no effect on p27. These results show that decorin is a signaling molecule in sprouting endothelial cells where it acts via different pathways, one of them involving Akt/protein kinase B.

Base Sequence↗
PubMed2001

Reactive carbonyl formation by oxidative and non-oxidative pathways.

The spectrophotometric protein carbonyl assay is used as an indicator of protein damage by free radical reactions in vitro and in a variety of pathologies. We investigated model proteins and a variety of oxidative and non-oxidative reactions, as well as what effects hemoglobin, myoglobin, and cytochrome c might have on levels of protein carbonyls. We show that oxidative as well as non-oxidative mechanisms introduce carbonyl groups into proteins, providing a moiety for quantification with 2,4-dinitrophenylhydrazine (DNPH). Bovine serum albumin exposed to oxidative scenarios, such as hypochlorous acid, peroxynitrite, and metal-catalyzed oxidation exhibited variable, but increased levels of carbonyls. Other non-oxidative modification systems, in which proteins are incubated with various aldehydes, such as malondialdehyde, acrolein, glycolaldehyde, and glyoxal also generated significant amounts of carbonyls. Furthermore, purified myoglobin, hemoglobin, and cytochrome c show high absorbance at the same wavelengths as DNPH. The high levels observed are due to the innate absorbance of hemoglobin, myoglobin, and cytochrome c near the assay spectra of DNPH. These studies show that carbonyl content could be due to oxidative as well as non-oxidative mechanisms and that heme-containing compounds may effect carbonyl quantification.

Aldehydes↗
PubMed2001

An inhibitory role of the phosphatidylinositol 3-kinase-signaling pathway in vascular endothelial growth factor-induced tissue factor expression.

Vascular endothelial growth factor (VEGF) is not only essential for vasculogenesis and angiogenesis but is also capable of inducing tissue factor, the prime initiator of coagulation, in endothelial cells. In this study we have analyzed the VEGF-elicited pathways involved in the induction of tissue factor in human umbilical cord vein endothelial cells. Using specific low molecular weight inhibitors we could demonstrate a crucial role of the p38 and Erk-1/2 mitogen-activated protein (MAP) kinases. In contrast, treatment with wortmannin or LY294002, inhibitors of phosphatidylinositol 3 (PI3)-kinase, resulted in a strong enhancement of the VEGF-induced tissue factor production, indicating a negative regulatory role of the PI3-kinase on tissue factor-inducing pathways. Accordingly, transduction with constitutively active Akt led to a reduction of VEGF-induced tissue factor production. Western blot analyses using antibodies specific for phosphorylated p38 showed an enhanced activation of this MAP kinase in human umbilical cord vein endothelial cells when stimulated with VEGF in the presence of wortmannin in comparison to either agent alone. Thus, the negative regulation of the PI3-kinase pathway on endothelial tissue factor activity can be explained at least in part by a suppression of this MAP kinase-signaling pathway. This is the first demonstration of a reciprocal relationship between procoagulant activity and the PI3-kinase-Akt signaling pathway, and it reveals a novel mechanism by which tissue factor expression can be controlled in endothelial cells.

Adenoviridae↗
PubMed2001

Adrenomedullin induces endothelium-dependent vasorelaxation via the phosphatidylinositol 3-kinase/Akt-dependent pathway in rat aorta.

To study the mechanisms by which adrenomedullin (AM) induces endothelium-dependent vasorelaxation, we examined whether AM-induced endothelium-dependent vasodilation was mediated by the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent pathway in rat aorta, because it was recently reported that PI3K/Akt was implicated in the activation of endothelial NO synthase. AM-induced vasorelaxation in thoracic aorta with intact endothelium was inhibited by pretreatment with PI3K inhibitors to the same level as that in endothelium-denuded aorta. AM elicited Akt phosphorylation in a time- and dose-dependent manner. AM-induced Akt phosphorylation was inhibited by pretreatment with a calmodulin-dependent protein kinase inhibitor as well as with PI3K inhibitors. When an adenovirus construct expressing a dominant-negative Akt mutant (Ad/dnAkt) was injected into abdominal aortas so that the mutant was expressed predominantly in the endothelium layer, AM-induced vasodilation was diminished to the same level as that in endothelium-denuded aortas. Finally, AM-induced cGMP production, which was used as an indicator for NO production, was suppressed by PI3K inhibition or by Ad/dnAkt infection into the endothelium. These results suggested that AM induced Akt activation in the endothelium via the Ca(2+)/calmodulin-dependent pathway and that this was implicated in the production of NO, which in turn induced endothelium-dependent vasodilation in rat aorta.

Adrenomedullin↗
PubMed2001

Phosphatidylinositol 3-kinase/Akt signaling controls endothelial cell sensitivity to Fas-mediated apoptosis via regulation of FLICE-inhibitory protein (FLIP).

Fas is constitutively expressed on endothelial cells, but in contrast to smooth muscle and other cell types, endothelial cells are highly resistant to Fas-mediated apoptosis. In this study, we examined the role of the serine/threonine kinase Akt/PKB in controlling the sensitivity of endothelial cells to Fas-mediated apoptosis. Serum deprivation inhibited expression of the caspase-8 inhibitor FLICE-inhibitory protein (FLIP), which functions downstream from Fas. FLIP expression levels were restored when serum-depleted cells were treated with vascular endothelial growth factor. Treatment with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin or LY294002 or infection of the adenoviral construct expressing dominant-negative Akt (Adeno-dnAkt) also inhibited the expression of FLIP in endothelial cells, whereas the MEK inhibitor PD98059 had no effect. Conversely, adenovirus-mediated transfection of a constitutively-active Akt gene abolished the wortmannin- and LY294002-mediated downregulation of FLIP. Suppression of PI 3-kinase signaling sensitized endothelial cells to Fas-mediated apoptosis. Under conditions of suppressed PI 3-kinase signaling, restoration of FLIP expression reversed the induced sensitivity of endothelial cells to Fas-mediated apoptosis. These data suggest that inhibition of Fas-mediated apoptosis, via promotion of FLIP expression, is a mechanism through which Akt signaling can promote endothelial cell survival.

Androstadienes↗
PubMed2001

Fas ligand overexpression on allograft endothelium inhibits inflammatory cell infiltration and transplant-associated intimal hyperplasia.

Despite recent advances in immunosuppressive therapy, accelerated coronary atherosclerosis remains a major problem in the long-term survival of transplant recipients. Chronic graft vasculopathy is believed to result from recipient inflammatory responses, and it is characterized by early mononuclear cell infiltration of the transplanted vessel. Here we show that endothelial cells can be genetically modified to overexpress functional, cell-surface Fas ligand (FasL) by adenovirus-mediated gene transfer without undergoing self-destruction. In a rodent model of transplant graft vasculopathy, endothelial overexpression of FasL attenuated T cell and macrophage infiltration at 1 wk posttransplantation. These vessels also displayed reduced neointima formation at one and 2 mo posttransplantation. These results indicate that inhibition of the early inflammatory response to allografted vessels by endothelial cell-specific overexpression of FasL may have utility in the treatment of transplant arteriosclerosis.

Animals↗
PubMed2001

Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells.

Vascular endothelial growth factor (VEGF) utilizes a phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling pathway to protect endothelial cells from apoptotic death. Here we show that PI 3-kinase/Akt signaling promotes endothelial cell survival by inhibiting p38 mitogen-activated protein kinase (MAPK)-dependent apoptosis. Blockade of the PI 3-kinase or Akt pathways in conjunction with serum withdrawal stimulates p38-dependent apoptosis. Blockade of PI 3-kinase/Akt also led to enhanced VEGF activation of p38 and apoptosis. In this context, the pro-apoptotic effect of VEGF is attenuated by the p38 MAPK inhibitor SB203580. VEGF stimulation of endothelial cells or infection with an adenovirus expressing constitutively active Akt causes MEKK3 phosphorylation, which is associated with decreased MEKK3 kinase activity and down-regulation of MKK3/6 and p38 MAPK activation. Conversely, activation-deficient Akt decreases VEGF-stimulated MEKK3 phosphorylation and increases MKK/p38 activation. Activation of MKK3/6 is not dependent on Rac activation since dominant negative Rac does not decrease p38 activation triggered by inhibition of PI 3-kinase. Thus, cross-talk between the Akt and p38 MAPK pathways may regulate the level of cytoprotection versus apoptosis and is a new mechanism to explain the cytoprotective actions of Akt.

Adenoviridae↗
PubMed2001

Sphingosine 1-phosphate activates Akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells.

Sphingosine 1-phosphate (SPP) binds to members of the endothelial differentiation gene family (EDG) of receptors and leads to diverse signaling events including cell survival, growth, migration and differentiation. However, the mechanisms of how SPP activates these proangiogenic pathways are poorly understood. Here we show that SPP signals through the EDG-1 receptor to the heterotrimeric G protein G(i), leading to activation of the serine/threonine kinase Akt and phosphorylation of the Akt substrate, endothelial nitric-oxide synthase (eNOS). Inhibition of G(i) signaling, and phosphoinositide 3-kinase (PI 3-kinase) activity resulted in a decrease in SPP-induced endothelial cell chemotaxis. SPP also stimulates eNOS phosphorylation and NO release and these effects are also attenuated by inhibition of G(i) signaling, PI 3-kinase, and Akt. However, inhibition of NO production did not influence SPP-induced chemotaxis but effectively blocked the chemotactic actions of vascular endothelial growth factor. Thus, SPP signals through G(i) and PI 3-kinase leading to Akt activation and eNOS phosphorylation.

Animals↗
PubMed2001

Activated Akt protects the lung from oxidant-induced injury and delays death of mice.

Oxidant-induced injury to the lung causes extensive damage to lung epithelial cells. Impaired protection and repair of the lung epithelium can result in death. The serine-threonine kinase Akt has been implicated in inhibiting cell death induced by different stimuli including growth factor withdrawal, cell cycle discordance, DNA damage, and loss of cell adhesion in different cell types. However, the in vivo relevance of this prosurvival pathway has not been explored. Here we show that a constitutively active form of Akt introduced intratracheally into the lungs of mice by adenovirus gene transfer techniques protects mice from hyperoxic pulmonary damage and delays death of mice. This is the first demonstration of the in vivo protective function of Akt in the context of oxidant-induced lung injury.

Animals↗
PubMed2001

Glycoprotein 130 regulates cardiac myocyte survival in doxorubicin-induced apoptosis through phosphatidylinositol 3-kinase/Akt phosphorylation and Bcl-xL/caspase-3 interaction.

BACKGROUND: We recently reported that the activation of glycoprotein (gp) 130 by leukemia inhibitory factor (LIF) upregulates Bcl-xL and exerts antiapoptotic effects in cardiac myocytes. In addition, LIF induces activation of phosphatidylinositol (PI) 3-kinase and Akt, which are known to be required for cell survival. However, their regulatory roles in cell death remain unknown. METHODS AND RESULTS: We investigated the fate of these proteins and the cytoprotective effects of LIF on doxorubicin (DOX)-induced apoptosis in cultured neonatal rat cardiac myocytes. Myocyte apoptosis increased significantly in DOX-treated cells but was significantly reduced by LIF pretreatment. The kinase activities of PI 3-kinase and Akt declined below basal levels but were partially recovered with LIF. Moreover, DOX-induced caspase-3 activation and decrease in Bcl-xL abundance are completely inhibited by LIF and caspase inhibitor. LIF phosphorylates Bad through PI 3-kinase and reduces the heterodimerization of Bad with Bcl-xL. Adenovirus transfer of the constitutively active form of Akt to cardiac myocytes restored cardiac myocyte survival after DOX treatment. Conversely, the dominant-negative form of Akt inhibited LIF-induced increase in cell viability and suppression of caspase-9 activation. CONCLUSIONS: Activation of gp130 inhibits DOX-induced cell death in cardiac myocytes, resulting in the restoration of PI 3-kinase/Akt activities and in the inactivation of caspase-3, leading to facilitation of the protective function of Bcl-xL.

Animals↗
PubMed2001

Phosphatidylinositol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells.

The caspase-8 homologue FLICE-inhibitory protein (FLIP) functions as a caspase-8 dominant negative, blocking apoptosis induced by the oligomerization of the adapter protein FADD/MORT-1. FLIP expression correlates with resistance to apoptosis induced by various members of the tumor necrosis factor family such as TRAIL. Furthermore, forced expression of FLIP renders cells resistant to Fas-mediated apoptosis. Although FLIP expression is regulated primarily by MEK1 activity in activated T cells, the oncogenic signaling pathways that regulate FLIP expression in tumor cells are largely unknown. In this report, we examined the roles of the MAP kinase and phosphatidylinositol (PI) 3-kinase signaling pathways in the regulation of FLIP expression in tumor cells. We observed that the MEK1 inhibitor PD98059 reduced FLIP levels in only 2 of 11 tumor cell lines tested. In contrast, disruption of the PI 3-kinase pathway with the specific inhibitor LY294002 reduced Akt (protein kinase B) phosphorylation and the levels of FLIP protein and mRNA in all cell lines evaluated. The introduction of a dominant negative Akt adenoviral construct also consistently reduced FLIP expression as well as the phosphorylation of the Akt target glycogen synthase kinase-3. In addition, infection of the same cell lines with a constitutively active Akt adenovirus increased FLIP expression and the phosphorylation of GSK-3. These data add FLIP to the growing list of apoptosis inhibitors in which expression or function is regulated by the PI 3-kinase-Akt pathway.

Apoptosis↗
PubMed2001

Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies.

M. Zhang, D. Methot, V. Poppa, Y. Fujio, K. Walsh and C. E. Murry. Cardiomyocyte Grafting for Cardiac Repair: Graft Cell Death and Anti-Death Strategies. Journal of Molecular and Cellular Cardiology (2001) 33, 907-921. Recent studies indicate that cardiomyocyte grafting forms new myocardium in injured hearts. It is unknown, however, whether physiologically significant amounts of new myocardium can be generated. Pilot experiments showed that death of grafted rat neonatal cardiomyocytes limited formation of new myocardium after acute cryoinjury. Time-course studies showed that, at 30 min after grafting, only 1.8(+/-0.4)% of graft cells were TUNEL-positive. At 1 day, however, TUNEL indices increased to 32.1(+/-3.5)% and remained high at 4 days, averaging 9.8(+/-3.8)%. By 7 days, TUNEL decreased to 1.0(+/-0.2)%. Electron microscopy revealed that dead cells had features of both irreversible ischemic injury and apoptosis. To test whether ischemia contributed to poor graft survival, grafts were placed into vascularized 2-week-old cardiac granulation tissue or normal myocardium. TUNEL indices were reduced by 53% and 86%, respectively. Adenoviral infection of graft cells with the cytoprotective kinase Akt, or constitutively active Akt, reduced TUNEL indices by 31% and 40%, respectively, compared to beta -gal-transfected controls. Neither treatment reached statistical significance compared to untreated controls, however. Heat shock reduced cardiomyocyte death in vitro in response to serum deprivation, glucose depletion, and viral activation of the Fas death pathway. When cardiomyocytes were heat shocked prior to grafting, graft cell death in vivo was reduced by 54% at day 1. Therefore, high levels of cardiomyocyte death occur for at least 4 days after grafting into injured hearts, in large part due to ischemia. Death can be limited by activating the Akt pathway and even more effectively by heat shock prior to transplantation.

Adenoviridae↗
PubMed2001

Beta-amyloid peptide expression is sufficient for myotube death: implications for human inclusion body myopathy.

Inclusion body myositis (sIBM) is the most common disorder of skeletal muscle in aged humans. It shares biochemical features with Alzheimer's disease, including congophilic deposits, which are immunoreactive for beta-amyloid peptide (Abeta) and C'-terminal betaAPP epitopes. However, the etiology of myofiber loss and the role of intracellular Abeta in IBM is unknown. Here we report correlative evidence for apoptotic cell death in myofibers of IBM patients that exhibit pronounced Abeta deposition. HSV-1-mediated gene transfer of Abeta(42) into cultured C2C12 myotubes resulted in a 12.6-fold increase in dUTP-labeled and condensed nuclei over nonexpressing myotubes (P < 0.05). The C'-terminal betaAPP domain C99 also induced myotube apoptosis, but to a significantly lesser extent than Abeta. Apoptosis specific to Abeta-expressing myotubes was also demonstrated through DNA fragmentation, decreased mitochondrial function and the loss of membrane phospholipid polarity. Myotubes laden with Abeta(42), but not other transgene products, developed cytoplasmic inclusions consisting of fibrillar material. Furthermore, injection of normal mouse gastrocnemius muscle with HSV-encoding Abeta cDNA resulted in TUNEL-positive myofibers with pyknotic nuclei. We conclude that Abeta is sufficient to induce apoptosis in myofibers both in vivo and in vitro and suggest it may contribute to myofiber loss and muscle dysfunction in patients with IBM.

Amyloid beta-Peptides↗
PubMed2001

Transcriptional regulation of vascular smooth muscle cell phenotype.

Vascular smooth muscle cell (VSMCs) modulate their state of differentiation in response to injury, and this plays an important role in proliferation and vascular regeneration. A number of transcription factors have been identified that function to coordinate VSMC differentiation and proliferation. Among them, GATA-6 and gax appear to favor the quiescent, differentiated state. On the other hand, BTEB2 and Egr-1 are associated with proliferative phenotype and factors such as SRF and MEF2 may have dual roles contributing to gene activation in both quiescent and proliferative VSMCs. In this paper, we will review the role of these transcription factors in VSMC differentiation and proliferation.

Animals↗