Biomedical subjects

Edward R Block

Publications and source records attributed to Edward R Block.

25 records · Page 2Linked to original sources

PubMed2002

Enhanced apoptosis in prolonged cultures of senescent porcine pulmonary artery endothelial cells.

Senescent or aged endothelial cells in culture remain metabolically active after cessation of division, and are generally believed to eventually die. However, mechanisms underlying the terminal aging of cultured cells, i.e. from senescence to death, are poorly understood. Here, we report that culturing of replicative senescent endothelial cells for a prolonged period of time without passaging leads to enhanced programmed cell death or apoptosis. Senescent (passage 45) and young (passage 3) porcine pulmonary artery endothelial cells (PAEC) were cultured for 0-42 days post confluence. The cells attached to culture dishes and floating in medium were collected at 0, 7, 14, 21, 28, 35 and 42 days post confluence and were assessed for markers of apoptosis. Morphology studies showed that ratios between senescent and young cells attached to dishes declined to 45% after 42 days postconfluence. Apoptotic cells in prolonged cultures of senescent PAEC increased from 5 to 35% as determined by protein mass, DNA breakage, and caspase-3 activation. Steady state levels of Bcl-2, an anti-apoptotic protein, in senescent prolonged cultures decreased to less than 20% for all time points compared with young cells. Relative levels of Bad, a pro-apoptotic protein, in senescent cells were elevated from 60 to 130% during prolonged culturing. These results indicate that terminal cellular aging enhances apoptosis and the levels of Bcl-2/Bad may be associated with the apoptotic process in porcine lung endothelial cells.

Animals↗

PubMed2002

Down-regulation of mitochondrial cytochrome c oxidase in senescent porcine pulmonary artery endothelial cells.

Cellular aging is associated with dysfunction of the mitochondrial respiration chain. Deficiency of mitochondrial cytochrome c oxidase (complex IV) plays a critical role in aging-induced mitochondrial dysfunction. We investigated whether in vitro cellular aging causes the downregulation of complex IV activity and gene expression using senescent (passage 45) and young (passage 3) pulmonary artery endothelial cells (PAEC). In senescent PAEC, the catalytic activity of complex IV decreased 84%, compared to that in young cells. Relative protein levels of complex IV subunits I and IV (complex IV S1 and S4) in senescent cells decreased 91%, compared to those in young cells. This suggests that lack of complex IV S1 and S4 in senescent cells may contribute to the deficiency of complex IV. Total steady state levels of mRNA for complex IV S1 and S4 in senescent cells were decreased to 20% and 18% of those in young cells. The relative rates of mRNA synthesis of complex IV S1 and S4 were decreased 46% and 37% in senescent cells, respectively, compared to young cells. The degradation of complex IV S1 and S4 was increased 76% and 64% in senescent cells, compared to young cells. These data indicate that mitochondrial DNA-encoded subunit I and nuclear DNA-encoded subunit IV of complex IV are downregulated through reduced synthesis and enhanced degradation of their mRNA, which may be responsible for the deficiency of complex IV in replicative senescent PAEC.

Animals↗

PubMed2002

Metabolism of dynorphins by peptidases of pulmonary artery endothelial cells.

Degradation of several dynorphins by peptidases expressed in cultured porcine pulmonary artery endothelial cells was studied by incubation of the peptide in cell suspensions followed by electrospray ionization and tandem mass spectrometric analyses. Under the in vitro conditions applied, only the metabolism of dynorphin A1-8 occurred in a significant extent. Studies involving specific peptidase inhibitors indicated that mainly bestatin-sensitive aminopeptidases, thiorphan-sensitive endopeptidases, and cFPAAF-pAB-sensitive endopeptidases expressed by the endothelial cells were involved in the process that converted dynorphin A1-8 to dynorphin A2-8, dynorphin A1-6, and leucine enkephalin (dynorphin A1-5), respectively. These peptidases may form a metabolic barrier for the cellular penetration of intact dynorphin A1-8 and/or control effects of the circulating peptide on endothelial opioid receptors of the cells.

Animals↗

PubMed2002

NO upregulation of a cyclic nucleotide-gated channel contributes to calcium elevation in endothelial cells.

We investigated whether nitric oxide (NO) upregulates a cyclic nucleotide-gated (CNG) channel and whether this contributes to sustained elevation of intracellular calcium levels ([Ca(2+)](i)) in porcine pulmonary artery endothelial cells (PAEC). Exposure of PAEC to an NO donor, NOC-18 (1 mM), for 18 h increased the protein and mRNA levels of CNGA2 40 and 50%, respectively (P < 0.05). [Ca(2+)](i) in NO-treated cells was increased 50%, and this increase was maintained for up to 12 h after removal of NOC-18 from medium. Extracellular calcium is required for the increase in [Ca(2+)](i) in NO-treated cells. Thapsigargin induced a rapid cytosolic calcium rise, whereas both a CNG and a nonselective cation channel blocker caused a faster decline in [Ca(2+)](i), suggesting that capacitive calcium entry contributes to the elevated calcium levels. Antisense inhibition of CNGA2 expression attenuated the NO-induced increases in CNGA2 expression and [Ca(2+)](i) and in capacitive calcium entry. Our results demonstrate that exogenous NO upregulates CNGA2 expression and that this is associated with elevated [Ca(2+)](i) and capacitive calcium entry in porcine PAEC.

Animals↗

PubMed2002

Activation of multiple signaling modules is critical in angiotensin IV-induced lung endothelial cell proliferation.

Signaling events involving angiotensin IV (ANG IV)-mediated pulmonary artery endothelial cell (PAEC) proliferation were examined. ANG IV significantly increased upstream phosphatidylinositide (PI) 3-kinase (PI3K), PI-dependent kinase-1 (PDK-1), extracellular signal-related kinases (ERK1/2), and protein kinase B-alpha/Akt (PKB-alpha) activities, as well as downstream p70 ribosomal S6 kinase (p70S6K) activities and/or phosphorylation of these proteins. ANG IV also significantly increased 5-bromo-2'-deoxy-uridine incorporation into newly synthesized DNA in a concentration- and time-dependent manner. Pretreatment of cells with wortmannin and LY-294002, inhibitors of PI3K, or rapamycin, an inhibitor of the mammalian target of rapamycin kinase and p70S6K, diminished the ANG IV-mediated activation of PDK-1 and PKB-alpha as well as phosphorylation of p70S6K. Although an inhibitor of mitogen-activated protein kinase kinase, PD-98059, but not rapamycin, blocked ANG IV-induced phosphorylation of ERK1/2, both PD-98059 and rapamycin independently caused partial reduction in ANG IV-mediated cell proliferation. However, simultaneous treatment with PD-98059 and rapamycin resulted in total inhibition of ANG IV-induced cell proliferation. These results demonstrate that ANG IV-induced DNA synthesis is regulated in a coordinated fashion involving multiple signaling modules in PAEC.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2002

Microtubule-active agents modify nitric oxide production in pulmonary artery endothelial cells.

The effects of specific microtubule-active agents on nitric oxide (NO) production were examined in pulmonary artery endothelial cells (PAEC). PAEC were incubated with taxol, which stabilizes microtubules, or nocodazole, which disrupts microtubules, or both for 2-4 h. We then examined NO production, endothelial NO synthase (eNOS) activity, and eNOS association with heat shock protein (HSP) 90. Incubation of PAEC with taxol (15 microM) for 2-4 h resulted in an increase in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. Incubation of PAEC with nocodazole (50 microM) for 2-4 h induced a decrease in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. The presence of taxol in the culture medium prevented the effects of nocodazole on NO production and eNOS activity in PAEC. Geldanamycin, a HSP90 inhibitor, prevented the taxol-induced increase in eNOS activity. Taxol and nocodazole did not affect eNOS, HSP90, and tubulin protein contents in PAEC, as detected using Western blot analysis. These results indicate that the polymerization state of the microtubule cytoskeleton regulates NO production and eNOS activity in PAEC. The changes in eNOS activity induced by modification of microtubules are due, at least in part, to the altered binding of HSP90 to eNOS protein.

Animals↗

PubMedDate not supplied

Senescence-enhanced oxidative stress is associated with deficiency of mitochondrial cytochrome c oxidase in vascular endothelial cells.

Cellular senescence-elevated oxidative stress plays a critical role in age-associated vascular endothelial dysfunction. We investigated whether deficiency of mitochondrial cytochrome c oxidase (complex IV) is causally linked to increased oxidant generation during cellular aging using senescent (passage 45) and young (passage 3) pulmonary artery endothelial cells (PAEC). In senescent PAEC, levels of O2- and H2O2 were elevated onefold, respectively, compared to those in young cells. Lipid peroxidation and protein carbonyl contents in aged cells were increased more than twofold compared to young cells. To determine whether lack of complex IV in senescent cells contributed to the increased oxidant generation, complex IV activity in young cells was specifically inhibited using antisense oligonucleotides directed against the mRNA of complex IV subunits. Levels of O2- and H2O2 in PAEC treated with antisense oligonucleotides were elevated onefold, respectively, which correlated with a similar increase in lipid (110%) and protein (20%) oxidation, compared to control oligonucleotides-transfected cells. Moreover, levels of nitrosylated proteins in antisense-transfected cells were increased 30%, compared to controls. These data demonstrate that deficiency of complex IV in senescent cells enhances oxidative and nitrosative stress, which may be responsible for senescence-induced endothelial cell loss and dysfunction.

Aging↗