Egr-1: is it always immediate and early?
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Animals↗
Biomedical subjects
D M Stern
Publications and source records attributed to D M Stern.
Stress-associated endoplasmic reticulum protein 1 (SERP1)/Ribosome-associated membrane protein 4 (RAMP4) stabilizes membrane proteins during stress and facilitates subsequent glycosylation.
Application of differential display to cultured rat astrocytes subjected to hypoxia allowed cloning of a novel cDNA, termed stress-associated endoplasmic reticulum protein 1 (SERP1). Expression of SERP1 was enhanced in vitro by hypoxia and/or reoxygenation or other forms of stress, causing accumulation of unfolded proteins in endoplasmic reticulum (ER) stress, and in vivo by middle cerebral artery occlusion in rats. The SERP1 cDNA encodes a 66-amino acid polypeptide which was found to be identical to ribosome-associated membrane protein 4 (RAMP4) and bearing 29% identity to yeast suppressor of SecY 6 protein (YSY6p), suggesting participation in pathways controlling membrane protein biogenesis at ER. In cultured 293 cells subjected to ER stress, overexpression of SERP1/RAMP4 suppressed aggregation and/or degradation of newly synthesized integral membrane proteins, and subsequently, facilitated their glycosylation when the stress was removed. SERP1/RAMP4 interacted with Sec61alpha and Sec61beta, which are subunits of translocon, and a molecular chaperon calnexin. Furthermore, Sec61alpha and Sec61beta, but not SERP1/RAMP4, were found to associate with newly synthesized integral membrane proteins under stress. These results suggest that stabilization of membrane proteins in response to stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, other components of translocon, and molecular chaperons in ER.
Amino Acid Sequence↗
Hypoxia-associated induction of early growth response-1 gene expression.
The paradigm for the response to hypoxia is erythropoietin gene expression; activation of hypoxia-inducible factor-1 (HIF-1) results in erythropoietin production. Previously, we found that oxygen deprivation induced tissue factor, especially in mononuclear phagocytes, by an early growth response (Egr-1)-dependent pathway without involvement of HIF-1 (Yan, S.-F., Zou, Y.-S., Gao, Y., Zhai, C., Mackman, N., Lee, S., Milbrandt, J., Pinsky, D., Kisiel, W., and Stern, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8298-8303). Now, we show that cultured monocytes subjected to hypoxia (pO2 approximately 12 torr) displayed increased Egr-1 expression because of de novo biosynthesis, with a approximately 10-fold increased rate of transcription. Transfection of monocytes with Egr-1 promoter-luciferase constructs localized elements responsible for hypoxia-enhanced expression to -424/-65, a region including EBS (ets binding site)-SRE (serum response element)-EBS and SRE-EBS-SRE sites. Further studies with each of these regions ligated to the basal thymidine kinase promoter and luciferase demonstrated that EBS sites in the element spanning -424/-375 were critical for hypoxia-enhanceable gene expression. These data suggested that an activated ets factor, such as Elk-1, in complex with serum response factor, was the likely proximal trigger of Egr-1 transcription. Indeed, hypoxia induced activation of Elk-1, and suppression of Elk-1 blocked up-regulation of Egr-1 transcription. The signaling cascade preceding Elk-1 activation in response to oxygen deprivation was traced to activation of protein kinase C-betaII, Raf, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase and mitogen-activated protein kinases. Comparable hypoxia-mediated Egr-1 induction and activation were observed in cultured hepatoma-derived cells deficient in HIF-1beta and wild-type hepatoma cells, indicating that the HIF-1 and Egr-1 pathways are initiated independently in response to oxygen deprivation. We propose that activation of Egr-1 in response to hypoxia induces a different facet of the adaptive response than HIF-1, one component of which causes expression of tissue factor, resulting in fibrin deposition.
Animals↗
150-kDa oxygen-regulated protein (ORP150) suppresses hypoxia-induced apoptotic cell death.
To determine the contribution of 150-kDa oxygen-regulated protein (ORP150) to cellular processes underlying adaptation to hypoxia, a cell line stably transfected to overexpress ORP150 antisense RNA was created. In human embryonic kidney (HEK) cells stably overexpressing ORP150 antisense RNA, ORP150 antigen and transcripts were suppressed to low levels in normoxia and hypoxia, whereas wild-type cells showed induction of ORP150 with oxygen deprivation. Inhibition of ORP150 in antisense transfectants was selective, as hypoxia-mediated enhancement of glucose-regulated protein (GRP) 78 and GRP94 was maintained. However, antisense ORP150 transfectants displayed reduced viability when subjected to hypoxia, compared with wild-type and sense-transfected HEK cells. In contrast, diminished levels of ORP150 had no effect on cytotoxicity induced by other stimuli, including oxygen-free radicals and sodium arsenate. Although cellular ATP content was similar in hypoxia, compared with ORP150 antisense transfectants and wild-type HEK cells, suppression of ORP150 expression was associated with accelerated apoptosis. Hypoxia-mediated cell death in antisense HEK transfectants did not cause an increase in caspase activity or in cytoplasmic cytochrome c antigen. A well recognized inducer of apoptosis in HEK cells, staurosporine, caused increased caspase activity and cytoplasmic cytochrome c levels in both wild-type and antisense cells. These data indicate that ORP150 has an important cytoprotective role in hypoxia-induced cellular perturbation and that ORP150-associated inhibition of apoptosis may involve mechanisms distinct from those triggered by other apoptotic stimuli.
Apoptosis↗
Role of ERAB/L-3-hydroxyacyl-coenzyme A dehydrogenase type II activity in Abeta-induced cytotoxicity.
Endoplasmic reticulum-associated amyloid beta-peptide (Abeta)-binding protein (ERAB)/L-3-hydroxyacyl-CoA dehydrogenase type II (HADH II) is expressed at high levels in Alzheimer's disease (AD)-affected brain, binds Abeta, and contributes to Abeta-induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a Km of approximately 68 microM and a Vmax of approximately 430 micromol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Abeta-mediated cellular dysfunction was studied by site-directed mutagenesis in the catalytic domain (Y168G/K172G). Although COS cells cotransfected to overexpress wild-type ERAB/HADH II and variant beta-amyloid precursor protein (betaAPP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and betaAPP(V717G) was without effect. We thus asked whether the enzyme might recognize alcohol substrates of which the aldehyde products could be cytotoxic; ERAB/HADH II catalyzed oxidation of a variety of simple alcohols (C2-C10) to their respective aldehydes in the presence of NAD+ and NAD-dependent oxidation of 17beta-estradiol. Addition of micromolar levels of synthetic Abeta(1-40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (Ki approximately 1.6 microM), as well as oxidation of 17beta-estradiol (Ki approximately 3.2 microM) and (-)-2-octanol (Ki approximately 2.6 microM). Because micromolar levels of Abeta were required to inhibit ERAB/HADH II activity, whereas Abeta binding to ERAB/HADH II occurred at much lower concentrations (Km approximately 40-70 nM), the latter more closely simulating Abeta levels within cells, Abeta perturbation of ERAB/HADH II was likely to result from mechanisms other than the direct modulation of enzymatic activity. Cells cotransfected to overexpress ERAB/HADH II and betaAPP(V717G) generated malondialdehyde-protein and 4-hydroxynonenal-protein epitopes, which were detectable only at the lowest levels in cells overexpressing either ERAB/HADH II or betaAPP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and betaAPP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Abeta-rich environment.
3-Hydroxyacyl CoA Dehydrogenases↗
Cellular cofactors for amyloid beta-peptide-induced cell stress. Moving from cell culture to in vivo.
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Alzheimer Disease↗
Interaction of baboon anti-alpha-galactosyl antibody with pig tissues.
As barriers to xenotransplantation are surmounted, such as suppression of hyperacute rejection allowing improved graft survival, it becomes important to define longer-term host-xenograft interactions. To this end we have prepared in baboons high titer anti-alpha-Galactosyl (alphaGal) and anti-porcine aortic endothelial cell antibodies, similar to human natural xenoantibodies and reactive with epitopes of thyroglobulin, laminin, and heparan sulfate proteoglycans. When injected into pigs with a protocol similar to that used in the rat to show the nephritogenic potential of heterologous anti-laminin and anti-heparan sulfate proteoglycan antibodies, baboon immunoglobulins bound first to renal vascular endothelium, and later to interstitial cells, especially fibroblasts and macrophages, and to antigens in basement membranes and extracellular matrix, where they colocalized with laminin- and heparan sulfate proteoglycan-antibodies, and with bound Griffonia simplicifolia B4. A similar binding was observed in other organs. The pigs did not develop an acute complement-dependent inflammation, but rather chronic lesions of the basement membranes and the extracellular matrix. Incubation of renal fibroblasts with baboon anti-alpha-Galactosyl antibodies resulted in increased synthesis of transforming growth factor-beta and collagen, suggesting a possible basis for the fibrotic response. The results demonstrate that in this experimental model a consequence of alphaGal antibody interaction with porcine tissues, is immunoreactivity with alphaGal on matrix molecules and interstitial cells, priming mechanisms leading to fibrosis resembling that in chronic allograft rejection. The possibility that similar lesions may develop in long-surviving pig xenografts is discussed.
Animals↗
Time-dependent cellular population of textured-surface left ventricular assist devices contributes to the development of a biphasic systemic procoagulant response.
OBJECTIVE: Textured-surface left ventricular assist devices (LVAD) have been shown to enhance ventricular function and survival in patients with end-stage heart failure. Furthermore, we have described a procoagulant physiology in our LVAD population with sustained thrombin generation (elevated thrombin-antithrombin III complex and prothrombin fragment 1+2) and fibrinolysis (D-dimers), even up to 335 days after LVAD placement. To explain such sustained activation of coagulation, we speculated that the LVAD surface selectively adsorbed and promoted activation of circulating blood cells. METHODS: In a prospective study of 20 patients with LVADs, we examined samples of peripheral blood as well as cells harvested from the surface of the LVADs at the time of their explantation for procoagulant proinflammatory markers. RESULTS: Analysis of the cells populating the LVAD surface revealed the presence of pluripotent hematopoietic CD34(+) cells, as well as cells bearing monocyte (CD14)/macrophage (CD68) markers, which also expressed procoagulant tissue factor. Reverse transcriptase-polymerase chain reaction confirmed cellular activation on the LVAD surface, revealing transcripts for interleukin 1alpha, interleukin 2, and tumor necrosis factor alpha, in addition to vascular cell adhesion molecule-1 consistent with their capacity to continually recruit and activate circulating cells, thereby propagating their response. In the periphery, elevated levels of tissue factor were found in the plasma of patients with LVADs, along with enhanced procoagulant activity. CONCLUSION: These observations suggest that the LVAD surface selectively absorbs and activates circulating hematopoietic precursor and monocytic cells, thereby creating a sustained prothrombotic and potentially proinflammatory systemic environment.
Adolescent↗
Alpha-galactosyl antibody redistributes alpha-galactosyl at the surface of pig blood and endothelial cells.
The interaction of antibodies with cell surface antigens may induce redistribution of immune complexes, followed by antigen depletion, with increased resistance to injurious effect of antibody and complement (antigenic modulation). Human natural antibodies to Gal alpha 1,3Gal beta 1,4GlcNAc-R (alpha Gal) epitopes expressed at the surface of pig cells are a major obstacle to xenotransplantation. Recent studies have shown that these antibodies do not modulate alpha Gal, but the morphological consequences of the antigen-antibody interaction are unknown. Pig blood and endothelial cells, were exposed to baboon alpha-Gal antibodies, and studied by immunofluorescence and phase contrast microscopy, flow cytometry, and inhibition enzyme-linked immunosorbent assay. In cells studied at 4 degrees C or fixed, alpha Gal was diffusely expressed at the surface. After cross-linking at 37 degrees C, antigenic modulation did not occur, but granular redistribution of alpha Gal immune complexes was seen in all cell types. In other systems a similar redistribution is known to induce perturbation of the plasma membrane/cytoskeletal structure with changes in adhesive properties, gene regulation, and T cell activation, which could be important if pig xenografts will be made to survive for prolonged periods.
Animals↗
Human endothelium: endovascular biopsy and molecular analysis.
PURPOSE: To develop a safe and reproducible method for harvesting viable vascular endothelium to analyze gene expression at sites of vascular lesions. MATERIALS AND METHODS: Coaxial curved stainless-steel guide wires were used to obtain samples of endothelial cells from large arteries and veins in 29 patients undergoing routine endovascular procedures. Three immunocytochemical markers were used to identify cells as endothelial. Cellular viability was evaluated in terms of cell membrane integrity, energy-dependent uptake of acetylated low-density lipoprotein, and cellular response to lipopolysaccharide. Single-cell reverse transcription polymerase chain reaction (PCR) and immunocytochemistry were used to study endothelial gene expression. RESULTS: Cells with endothelial morphology and immunoreactivity for von Willebrand factor, thrombomodulin, and angiotensin-converting enzyme were consistently obtained from iliac and carotid arteries and large veins (average yield [+/- standard error] from 26 iliac arteries, 262 endothelial cells +/- 45, 20%-30% of which were viable). These cells displayed induction of E-selection messenger RNA at PCR after exposure to lipopolysaccharide. Expression of vascular cell adhesion molecule 1 transcripts in endothelial cells increased with patient age (P < .01), whereas expression of intercellular adhesion molecule 1 did not. CONCLUSION: Viable endothelium can be obtained during routine angiography. Immunocytochemical and reverse transcription PCR analyses of these cells allowed determination of transcripts and proteins expressed by endothelium at sites of vascular lesions. Such information could aid in understanding mechanisms of vascular diseases and in clinical decision making.
Adolescent↗
Hypoxia/Hypoxemia-Induced activation of the procoagulant pathways and the pathogenesis of ischemia-associated thrombosis.
Although oxygen deprivation has long been associated with triggering of the procoagulant pathway and venous thrombosis, blood hypoxemia and stasis by themselves do not lead to fibrin formation. A pathway is outlined through which diminished levels of oxygen activate the transcription factor early growth response-1 (Egr-1) leading to de novo transcription/translation of tissue factor in mononuclear phagocytes and smooth muscle cells, which eventuates in vascular fibrin deposition. The procoagulant response is magnified by concomitant suppression of fibrinolysis by hypoxia-mediated upregulation of plasminogen activator inhibitor-1. These data add a new facet to the biology of thrombosis associated with hypoxemia/stasis and imply that interference with mechanisms causing Egr-1 activation in response to oxygen deprivation might prevent vascular fibrin deposition occurring in ischemia without directly interfering with other pro/anticoagulant pathways.
Animals↗
Membranous glomerulonephritis induced in the pig by antibody to angiotensin-converting enzyme: considerations on its relevance to the pathogenesis of human idiopathic membranous glomerulonephritis.
In the course of studies on the humoral consequences of swine to primate xenotransplantation, the investigators induced formation of glomerular subepithelial immune deposits and tubular lesions in pigs injected with heterologous antibody to angiotensin-converting enzyme. This study describes the morphology of the lesions, discusses their mechanism, explains their relevance for understanding the pathogenesis of human idiopathic membranous glomerulonephritis, and proposes future directions for investigations.
Animals↗
Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition.
Oxygen deprivation, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. To investigate the effects of low ambient oxygen tension on the fibrinolytic system, mice were placed in a hypoxic environment with pO2 < 40 Torr. Plasma levels of plasminogen activator inhibitor-1 (PAI-1) antigen, detected by ELISA, increased in a time-dependent fashion after hypoxic exposure (increased as early as 4 h, P < 0.05 vs. normoxic controls), and were accompanied by an increase in plasma PAI-1 activity by 4 h (P < 0.05 vs. normoxic controls). Northern analysis of hypoxic murine lung demonstrated an increase in PAI-1 mRNA compared with normoxic controls; in contrast, transcripts for both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) decreased under hypoxic conditions. Immunocolocalization studies identified macrophages as the predominant source of increased PAI-1 within hypoxic lung. Using a transformed murine macrophage line, striking induction of PAI-1 transcripts occurred under hypoxic conditions, due to both increased de novo transcription as well as increased mRNA stability. Consistent with an important role of the fibrinolytic system in hypoxia-induced fibrin accumulation, PAI-1 +/+ mice exposed to hypoxia exhibited increased pulmonary fibrin deposition based upon a fibrin immunoblot, intravascular fibrin identified by immunostaining, and increased accumulation of 125I-fibrinogen/fibrin in hypoxic tissue. In contrast, mice deficient for the PAI-1 gene (PAI-1 -/-) similarly exposed to hypoxic conditions did not display increased fibrin accumulation compared with normoxic PAI-1 +/+ controls. Furthermore, homozygous null uPA (uPA -/-) and tPA (tPA -/-) mice subjected to oxygen deprivation showed increased fibrin deposition compared with wild-type controls. These studies identify enhanced expression of PAI-1 as an important mechanism suppressing fibrinolysis under conditions of low oxygen tension, a response which may be further amplified by decreased expression of plasminogen activators. Taken together, these data provide insight into an important potential role of macrophages and the fibrinolytic system in ischemia-induced thrombosis.
Animals↗
Endothelial cells in physiology and in the pathophysiology of vascular disorders.
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Animals↗
Thrombomodulin modulates growth of tumor cells independent of its anticoagulant activity.
Thrombomodulin (TM), recognized as an essential vessel wall cofactor of the antithrombotic mechanism, is also expressed by a wide range of tumor cells. Tumor cell lines subcloned from four patients with malignant melanoma displayed a negative correlation between TM expression and cell proliferation in vitro and in vivo. Overexpression of wild-type TM decreased cell proliferation in vitro and tumor growth in vivo. TM mutants with altered protein C activation capacity lead to a similar effect. In contrast, transfection of melanoma cells with mutant TM constructs, in which a portion of the cytoplasmic or lectin domain was deleted, abrogated the antiproliferative effect associated with overexpression of wild-type TM. Experiments performed with either peptide agonists/antagonists of the thrombin receptor, with hirudin, or with inhibitors of thrombin-TM interaction did not alter the growth inhibitory effect of TM overexpression. These data suggest that TM exerts an effect on cell proliferation independent of thrombin and the thrombin receptor, possibly related to the binding of novel ligands to determinants in the lectin domain which might trigger signal transduction pathways dependent on the cytoplasmic domain.
Animals↗
Selective anticoagulation with active site-blocked factor IXA suggests separate roles for intrinsic and extrinsic coagulation pathways in cardiopulmonary bypass.
BACKGROUND: Multiple stimuli converge in cardiopulmonary bypass to create a tremendous prothrombotic stimulus. The ideal anticoagulant for cardiopulmonary bypass should selectively target only the intravascular stimuli, thereby eliminating pathologic clotting in the bypass circuit while preserving hemostasis in the thoracic cavity. We propose the inhibition of factor IX as such a targeted anticoagulant strategy. METHODS: We prepared an inhibitor of activated factor IX and applied it to a primate model of cardiopulmonary bypass to confirm the anticoagulant efficacy of activated factor IX in this setting and to assess more subtle markers of thrombin generation, macrophage procoagulant activity, and cellular tissue factor expression. Seven baboons that received activated factor IX (460 microg/kg) and 7 that received heparin (300 IU/kg) and protamine underwent cardiopulmonary bypass for 90 minutes and were followed after the operation for 3 hours. RESULTS: Analysis of plasma factor IX activity demonstrated adequate inhibition (<20%) of factor IX throughout cardiopulmonary bypass. Activated factor IX-treated baboons demonstrated similar circuit patency to heparin-treated baboons but had significantly diminished intraoperative blood loss. Preservation of extravascular hemostasis was further demonstrated in activated factor IX-treated animals by (1) significantly increased levels of thrombin-antithrombin III complex and prothrombin activation peptide (F1+2) without intravascular thrombosis, (2) significantly greater macrophage procoagulant activity in pericardial-derived monocytes, and (3) immunohistochemical evidence of tissue factor expression in pericardial mesothelial cells and macrophages. CONCLUSIONS: Anticoagulation with activated factor IX allows for intravascular anticoagulation with maintenance of extravascular hemostasis. These findings suggest activated factor IX as an agent that not only exemplifies a targeted approach to selective anticoagulation in cardiac surgery but also further characterizes the procoagulant milieu during cardiopulmonary bypass.
Animals↗
Heparinless cardiopulmonary bypass with active-site blocked factor IXa: a preliminary study on the dog.
OBJECTIVE: Cardiopulmonary bypass is a potent stimulus for activation of procoagulant pathways. Heparin, the traditional antithrombotic agent, however, is often associated with increased perioperative blood loss because of its multiple sites of action in the coagulation cascade and its antiplatelet and profibrinolytic effects. Furthermore, heparin-mediated immunologic reactions (that is, heparin-induced thrombocytopenia) may contraindicate its use. Cardiopulmonary bypass with a selective factor IXa inhibitor was tested to see whether it could effectively limit bypass circuit/intravascular space thrombosis while decreasing extravascular bleeding, thereby providing an alternative anticoagulant strategy when heparin may not be safely administered. METHODS: Active site-blocked factor IXa, a competitive inhibitor of the assembly of factor IXa into the factor X activation complex, was prepared by modification of the enzyme's active site by the use of dansyl glutamic acid-glycine-arginine-chlormethylketone. Twenty mongrel dogs (five were given standard heparin/protamine; 15 were given activated site-blocked factor IXa doses ranging from 300 to 600 microg/kg) underwent 1 hour of hypothermic cardiopulmonary bypass, and blood loss was monitored for 3 hours after the procedure. RESULTS: Use of activated site-blocked factor IXa as an anticoagulant in cardiopulmonary bypass limited fibrin deposition within the extracorporeal circuit as assessed by scanning electron microscopy, comparable with the antithrombotic effect seen with heparin. In contrast to heparin, effective antithrombotic doses of activated site-blocked factor IXa significantly diminished blood loss in the thoracic cavity and in an abdominal incisional bleeding model. CONCLUSION: These initial studies on the dog suggest that administration of activated site-blocked factor IXa may be an effective alternative anticoagulant strategy in cardiopulmonary bypass when heparin is contraindicated, affording inhibition of intravascular/extracorporeal circuit thrombosis with enhanced hemostasis in the surgical wound.
Animals↗
Hypoxia-mediated modulation of vascular function--implications for organ preservation and thrombogenesis: Roger S. Mitchell lecture.
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Animals↗