Translational silencing of ceruloplasmin requires the essential elements of mRNA circularization: poly(A) tail, poly(A)-binding protein, and eukaryotic translation initiation factor 4G.

Ceruloplasmin (Cp) is a glycoprotein secreted by the liver and monocytic cells and probably plays roles in inflammation and iron metabolism. We showed previously that gamma interferon (IFN-gamma) induced Cp synthesis by human U937 monocytic cells but that the synthesis was subsequently halted by a transcript-specific translational silencing mechanism involving the binding of a cytosolic factor(s) to the Cp mRNA 3' untranslated region (UTR). To investigate how protein interactions at the Cp 3'-UTR inhibit translation initiation at the distant 5' end, we considered the "closed-loop" model of mRNA translation. In this model, the transcript termini are brought together by interactions of poly(A)-binding protein (PABP) with both the poly(A) tail and initiation factor eIF4G. The effect of these elements on Cp translational control was tested using chimeric reporter transcripts in rabbit reticulocyte lysates. The requirement for poly(A) was shown since the cytosolic inhibitor from IFN-gamma-treated cells minimally inhibited the translation of a luciferase reporter upstream of the Cp 3'-UTR but almost completely blocked the translation of a transcript containing a poly(A) tail. Likewise, a requirement for poly(A) was shown for silencing of endogenous Cp mRNA. We considered the possibility that the cytosolic inhibitor blocked the interaction of PABP with the poly(A) tail or with eIF4G. We found that neither of these interactions were inhibited, as shown by immunoprecipitation of PABP followed by quantitation of the poly(A) tail by reverse transcription-PCR and of eIF4G by immunoblot analysis. We considered the alternate possibility that these interactions were required for translational silencing. When PABP was depleted from the reticulocyte lysate with anti-human PABP antibody, the cytosolic factor did not inhibit translation of the chimeric reporter, thus showing the requirement for PABP. Similarly, in lysates treated with anti-human eIF4G antibody, the cytosolic extract did not inhibit the translation of the chimeric reporter, thereby showing a requirement for eIF4G. These data show that translational silencing of Cp requires interactions of three essential elements of mRNA circularization, poly(A), PABP, and eIF4G. We suggest that Cp mRNA circularization brings the cytosolic Cp 3'-UTR-binding factor into the proximity of the translation initiation site, where it silences translation by an undetermined mechanism. These results suggest that in addition to its important function in increasing the efficiency of translation, transcript circularization may serve as an essential structural determinant for transcript-specific translational control.

Palmitoylation of caveolin-1 in endothelial cells is post-translational but irreversible.

Caveolin-1 is a palmitoylated protein involved in assembly of signaling molecules in plasma membrane subdomains termed caveolae and in intracellular cholesterol transport. Three cysteine residues in the C terminus of caveolin-1 are subject to palmitoylation, which is not necessary for caveolar targeting of caveolin-1. Protein palmitoylation is a post-translational and reversible modification that may be regulated and that in turn may regulate conformation, membrane association, protein-protein interactions, and intracellular localization of the target protein. We have undertaken a detailed analysis of [(3)H]palmitate incorporation into caveolin-1 in aortic endothelial cells. The linkage of palmitate to caveolin-1 was hydroxylamine-sensitive and thus presumably a thioester bond. However, contrary to expectations, palmitate incorporation was blocked completely by the protein synthesis inhibitors cycloheximide and puromycin. In parallel experiments to show specificity, palmitoylation of aortic endothelial cell-specific nitric-oxide synthase was unaffected by these reagents. Inhibitors of protein trafficking, brefeldin A and monensin, blocked caveolin-1 palmitoylation, indicating that the modification was not cotranslational but rather required caveolin-1 transport from the endoplasmic reticulum and Golgi to the plasma membrane. In addition, immunophilin chaperones that form complexes with caveolin-1, i.e. FK506-binding protein 52, cyclophilin A, and cyclophilin 40, were not necessary for caveolin-1 palmitoylation because agents that bind immunophilins did not inhibit palmitoylation. Pulse-chase experiments showed that caveolin-1 palmitoylation is essentially irreversible because the release of [(3)H]palmitate was not significant even after 24 h. These results show that [(3)H]palmitate incorporation is limited to newly synthesized caveolin-1, not because incorporation only occurs during synthesis but because the continuous presence of palmitate on caveolin-1 prevents subsequent repalmitoylation.

Regional differences in phorbol 12-myristate 13-acetate stimulation of PDGF production in the normal canine aorta.

OBJECTIVE: Platelet-derived growth factor (PDGF) is a potent smooth muscle cell mitogen implicated in the development of intimal hyperplasia and atherosclerosis. A regional variation in canine aortic production of PDGF (greater in the distal than in the proximal aorta) was demonstrated previously in organ culture. The response of aortic segments in organ culture, as well as of aortic endothelial cells and smooth muscle cells, to stimulators of PDGF secretion-phorbol 12-myristate 13-acetate (PMA) and thrombin-was assessed to elucidate whether these regional variations were due to intrinsic differences in the abilities of cells to produce PDGF. METHODS: Proximal and distal aortic segments were removed from 10 dogs and placed in organ culture, then treated with PMA or thrombin for 72 hours. PDGF in the conditioned media was measured by radioreceptor assay. RESULTS: PDGF production in the distal, unstimulated aorta was 2.5-fold higher than that in the proximal aorta (P <.05). Treatment of the proximal aorta with 10 nmol/L and 100 nmol/L PMA increased PDGF production twofold and threefold, respectively, whereas no increase with PMA treatment was seen in the distal aorta. After thrombin treatment, no increase in PDGF production was noted in the proximal aorta and only a minimal increase was noted in the distal aorta. Endothelial cells and smooth muscle cells (n = 6) were cultured from four aortic segments (ascending thoracic, descending thoracic, abdominal, and infrarenal) and treated with PMA. PDGF production by unstimulated endothelial cells from the infrarenal aorta was 2.5-fold higher (P <.01) than that from the ascending thoracic aorta. With PMA treatment, PDGF secretion increased in endothelial cells from all segments, the greatest percentage increase being observed in the proximal segments. Thrombin also increased PDGF release from endothelial cells, but with no regional variation. Unstimulated smooth muscle cells did not exhibit regional variation in PDGF production and did not increase PDGF secretion after treatment with PMA or thrombin. CONCLUSIONS: These findings suggest that endothelial cells in the aorta may have a differential capacity to produce PDGF in response to stimulants, reflecting intrinsic differences in endothelial cells from the proximal aorta versus the distal aorta, and this may account in part for the propensity of the distal aorta to develop atherosclerosis.

Ceruloplasmin and cardiovascular disease.

Transition metal ion-mediated oxidation is a commonly used model system for studies of the chemical, structural, and functional modifications of low-density lipoprotein (LDL). The physiological relevance of studies using free metal ions is unclear and has led to an exploration of free metal ion-independent mechanisms of oxidation. We and others have investigated the role of human ceruloplasmin (Cp) in oxidative processes because it the principal copper-containing protein in serum. There is an abundance of epidemiological data that suggests that serum Cp may be an important risk factor predicting myocardial infarction and cardiovascular disease. Biochemical studies have shown that Cp is a potent catalyst of LDL oxidation in vitro. The pro-oxidant activity of Cp requires an intact structure, and a single copper atom at the surface of the protein, near His(426), is required for LDL oxidation. Under conditions where inhibitory protein (such as albumin) is present, LDL oxidation by Cp is optimal in the presence of superoxide, which reduces the surface copper atom of Cp. Cultured vascular endothelial and smooth muscle cells also oxidize LDL in the presence of Cp. Superoxide release by these cells is a critical factor regulating the rate of oxidation. Cultured monocytic cells, when activated by zymosan, can oxidize LDL, but these cells are unique in their secretion of Cp. Inhibitor studies using Cp-specific antibodies and antisense oligonucleotides show that Cp is a major contributor to LDL oxidation by these cells. The role of Cp in lipoprotein oxidation and atherosclerotic lesion progression in vivo has not been directly assessed and is an important area for future studies.

The migratory response to platelet-derived growth factor of smooth muscle cells isolated from synthetic vascular grafts in a canine model.

OBJECTIVE: Previous studies on smooth muscle cells (SMCs) harvested from implanted synthetic grafts demonstrate increased production of platelet-derived growth factor (PDGF) but decreased proliferative response compared with aortic SMCs. The purpose of this study was to determine the migratory response of graft versus aortic SMCs. METHODS: Thoracoabdominal grafts were implanted in beagles. The SMCs were harvested from the graft and infrarenal aorta. Migration was determined with the use of a razor-scrape assay and computerized image analysis. RESULTS: The mean distance migrated and the number of cells that migrated were greater in graft SMCs at baseline (185 +/- 18 micrometer and 108 +/- 17 cells) compared with aortic cells (110 +/- 10 micrometer and 42 +/- 5 cells)(P <.05). Baseline differences persisted after treatment with antibodies to PDGF. The addition of PDGF (10 ng/mL) resulted in increased migration in both graft (229 +/- 23 micrometer and 146 +/- 20 cells) and aortic SMCs (130 +/- 9 micrometer and 70 +/- 5 cells) compared with baseline (P <.05). The relative increase in response to PDGF was similar between the two groups (P = not significant). CONCLUSIONS: Graft SMCs differ phenotypically from aortic SMCs; they exhibit increased basal migration that is independent of autocrine stimulation by PDGF. In contrast to their blunted proliferative response, graft SMCs have a similar migratory response to PDGF compared with aortic SMCs.

Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency.

A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these observations.

Mechanism of dacron-activated monocytic cell oxidation of low density lipoprotein.

PURPOSE: Oxidized lipids are believed to contribute to atherogenesis and may play a role in the development of anastomotic intimal hyperplasia in prosthetic vascular grafts. This study examines the hypothesis that clinically relevant graft material activates monocytes to oxidize low density lipoprotein (LDL). METHODS: LDL and Dacron or expanded polytetrafluoroethylene (ePTFE) graft material were incubated in the presence of U937 cells, a monocytic cell line. LDL oxidation was measured by conjugated dienes, lipid peroxides, thiobarbituric acid-reacting substances, and electrophoretic mobility. Cell production of superoxide was measured by ferricytochrome c reduction. Metal ion requirement was assessed with the metal chelators, ethylenediaminetetra-acidic acid, deferoxamine, and bathocuproinedisulfonic acid. To determine whether human monocytes were capable of being activated by Dacron graft material to oxidize LDL, freshly isolated peripheral blood monocytes were also studied. RESULTS: Incubation of LDL with U937 cells and Dacron increased LDL oxidation by 5- to 20-fold. LDL incubated with ePTFE or U937 cells alone resulted in minimal oxidation. Dacron graft increased U937 cell production of superoxide by 4-fold, whereas ePTFE had no effect. Superoxide dismutase inhibited Dacron-activated U937 cell oxidation of LDL by greater than 50%, which indicates a role for superoxide. Ethylenediaminetetra-acidic acid, deferoxamine, and bathocuproinedisulfonic acid each inhibited Dacron-activated U937 cell oxidation of LDL. Human peripheral blood monocytes were activated by Dacron graft material to oxidize LDL; superoxide dismutase inhibited Dacron-activated human monocytic oxidation of LDL, which suggests a role for superoxide. CONCLUSION: These results suggest that Dacron graft material activates monocytes to oxidize LDL by a mechanism that involves superoxide and requires iron and copper ions. Our work suggests a mechanism by which lipids that have been deposited within implanted vascular grafts may become oxidized. Oxidized lipids may contribute to the cellular dysfunction that results in anastomotic intimal hyperplasia and graft failure.

Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region.

Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine activation.

The proliferative response to platelet-derived growth factor of smooth muscle cells isolated from synthetic vascular grafts in a canine model.

OBJECTIVE: Previous studies on graft healing have shown increased platelet-derived growth factor (PDGF) production in graft segments versus native aortic segments. The purpose of this study was to characterize the proliferative response of graft smooth muscle cells (SMCs) to PDGF. METHODS: Thoracoabdominal grafts were implanted in beagles. SMCs were harvested from the graft and the proximal and distal aortas. Basal proliferation was assessed with growth curves in primary culture. The proliferative response to PDGF then was compared with [3H]thymidine uptake studies and cell counts. Finally, PDGF receptors were characterized with radio-labeled ligand binding assays. RESULTS: The growth curves showed that the graft SMCs entered log-phase growth 2 days earlier than did the aortic SMCs. Stimulation of quiescent early-passage graft SMCs with PDGF (10 ng/mL) resulted in a 1.7 +/- 0.1-fold increase in [3H]thymidine incorporation, which was significantly less than that of the SMCs from both the proximal aorta (11.8 +/- 3.0) and the distal aorta (10. 2 +/- 1.9; P <.5). Similarly, the 1.1 +/- 0.1-fold increase in graft SMC cell number was significantly less than the increases for both proximal (2.8 +/- 0.5) and distal (2.9 +/- 0.8) aortic SMCs (P <.5). Binding studies on quiescent first-passage cells showed fewer PDGF receptors available for binding in the graft SMCs (185 +/- 70 fmol/million cells) as compared with both the proximal (419 +/- 147 fmol/million cells) and the distal (387 +/- 112 fmol/million cells) aortas (P <.5). Binding affinity was similar for the three groups. CONCLUSION: Graft SMCs exist in a chronic proliferative state but exhibit a decreased proliferative response to PDGF and have fewer receptors available for binding PDGF than do aortic SMCs in vitro.

Prevalence and treatment of pain in older adults in nursing homes and other long-term care institutions: a systematic review.

BACKGROUND: The high prevalence of pain in older adults and its impact in this age group make it a public health issue, yet few studies of pain relief focus on older adults. Residents of long-term care facilities have more cognitive impairment than their community-living counterparts and may have difficulty reporting the presence and severity of pain. This systematic literature review was conducted to determine the prevalence of pain, and the type and effectiveness of interventions that have been used to treat pain in residents of nursing homes. METHODS: Studies were identified by searching MEDLINE (from January 1966 to May 1997), HEALTH (from January 1975 to May 1997), CINAHL (from January 1982 to April 1997), AGELINE (from January 1978 to April 1997) and the Cochrane Library (1997, issue 1) and by performing a manual search of textbooks and reference lists. Studies of any methodological design were included if they estimated the prevalence of pain in nursing homes or other long-term care institutions or evaluated interventions for the treatment of pain in residents. Of the 14 eligible studies, 12 were noncomparative studies, 1 was a comparison study with nonrandomized contemporaneous controls, and 1 was a randomized controlled trial. Information on several factors was extracted from each study, including study design, number of patients and facilities, main outcomes measured, methods used to identify and detect pain, prevalence and types of pain, and interventions used to treat pain. The strength of the evidence provided by each study was also assessed. RESULTS: In the 6 studies with data from self-reporting or chart reviews, the prevalence of pain ranged from 49% to 83%. In the 5 studies with data on analgesic use only, the prevalence of pain ranged from 27% to 44%. Only 3 studies, with just 30 patients in total, evaluated an intervention for the treatment of pain. INTERPRETATION: Despite the high prevalence of pain in residents of nursing homes, there is a lack of studies evaluating interventions to relieve their pain. The authors make recommendations for future studies in this area.

Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism.

The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates. A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of "aceruloplasminemic" patients who have extensive iron deposits in multiple tissues. We have recently shown that Cp increases iron uptake by cultured HepG2 cells. In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake. Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2-3-fold, using a transferrin receptor-independent pathway. Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter. Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations. These results indicate that Cp utilizes a trivalent cation-specific transporter. Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

Ceruloplasmin copper induces oxidant damage by a redox process utilizing cell-derived superoxide as reductant.

Oxidative damage by transition metals bound to proteins may be an important pathogenic mechanism. Ceruloplasmin (Cp) is a Cu-containing plasma protein thought to be involved in oxidative modification of lipoproteins. We have previously shown that Cp increased cell-mediated low-density lipoprotein (LDL) oxidation by a process requiring cell-derived superoxide, but the underlying chemical mechanism(s) is (are) unknown. We now show that superoxide reduction of Cp Cu is a critical reaction in cellular LDL oxidation. By bathocuproine disulfonate (BCS) binding and by superoxide utilization, we showed that exogenous superoxide reduces a single Cp Cu atom, the same Cu required for LDL oxidation. The Cu atom remained bound to Cp during the redox cycle. Three avenues of evidence showed that vascular cells reduce Cp Cu by a superoxide-dependent process. The 2-fold higher rate of Cp Cu reduction by smooth muscle cells (SMC) compared to endothelial cells (EC) was consistent with their relative rates of superoxide release. Furthermore, Cp Cu reduction by cells was blocked by Cu,Zn superoxide dismutase (SOD1). Finally, the level of superoxide produced by EC and SMC was sufficient to cause the amount of Cu reduction observed. An important role of Cp Cu reduction in LDL oxidation was suggested by results showing that SOD1 inhibited Cp Cu reduction and LDL oxidation by SMC with equal potency, while tumor necrosis factor-alpha stimulated both processes. In summary, these results show that superoxide is a critical cellular reductant of divalent transition metals involved in oxidation, and that protein-bound Cu is a substrate for this reaction. The role of these mechanisms in oxidative processes in vivo has yet to be defined.

Role of ceruloplasmin in cellular iron uptake.

Individuals with hereditary ceruloplasmin (Cp) deficiency have profound iron accumulation in most tissues, which suggests that Cp is important for normal release of cellular iron. Here, in contrast to expectations, Cp was shown to increase iron uptake by HepG2 cells, increasing the apparent affinity for the substrate by three times. Consistent with its role in iron uptake, Cp synthesis was regulated by iron supply and was increased four- to fivefold after iron depletion. Unlike other iron controllers that are posttranscriptionally regulated, Cp synthesis was transcriptionally regulated. Thus, iron-deficient cells could increase Cp synthesis to maintain intracellular iron homeostasis, so that defects would lead to global accumulation of iron in tissues.

Identification of the prooxidant site of human ceruloplasmin: a model for oxidative damage by copper bound to protein surfaces.

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.

Induction of ceruloplasmin synthesis by IFN-gamma in human monocytic cells.

Ceruloplasmin is a 132-kDa glycoprotein abundant in human plasma. It has multiple in vitro activities, including copper transport, lipid pro- and antioxidant activity, and oxidation of ferrous ion and aromatic amines; however, its physiologic role is uncertain. Although ceruloplasmin is synthesized primarily by the liver in adult humans, production by cells of monocytic origin has been reported. We here show that IFN-gamma is a potent inducer of ceruloplasmin synthesis by monocytic cells. Activation of human monoblastic leukemia U937 cells with IFN-gamma increased the production of ceruloplasmin by at least 20-fold. The identity of the protein was confirmed by plasmin fingerprinting. IFN-gamma also increased ceruloplasmin mRNA. Induction followed a 2- to 4-h lag and was partially blocked by cycloheximide, indicating a requirement for newly synthesized factors. Ceruloplasmin induction in monocytic cells was agonist specific, as IL-1, IL-4, IL-6, IFN-alpha, IFN-beta, TNF-alpha, and LPS were completely ineffective. The induction was also cell type specific, as IFN-gamma did not induce ceruloplasmin synthesis in endothelial or smooth muscle cells. In contrast, IFN-gamma was stimulatory in other monocytic cells, including THP-1 cells and human peripheral blood monocytes, and also in HepG2 cells. Ceruloplasmin secreted by IFN-gamma-stimulated U937 cells had ferroxidase activity and was, in fact, the only secreted protein with this activity. Monocytic cell-derived ceruloplasmin may contribute to defense responses via its ferroxidase activity, which may drive iron homeostasis in a direction unfavorable to invasive organisms.

Platelet-derived growth factor production by cells from Dacron grafts implanted in a canine model.

PURPOSE: Previous studies of grafts implanted in dogs documented a time-dependent increase in platelet-derived growth factor (PDGF) production that correlated with inner-capsule thickness. The purpose of this study was to identify the cells in vascular grafts that produce PDGF. METHODS: Dacron thoracoabdominal grafts were seeded with autologous endothelial cells (ECs), implanted in 11 beagles, and removed after 4 or 20 weeks. ECs and smooth muscle cells (SMCs) were cultured from grafts and adjacent aorta, and PDGF in the conditioned media was measured by radioreceptor assay. The PDGF A-chain mRNA level in freshly harvested cells was assessed using reverse transcriptase, followed by polymerase chain reaction, and expressed as a ratio of glyceraldehyde-3-phosphate dehydrogenase signal. Localization of PDGF A-chain and B-chain protein was also examined with immunohistochemical analysis. RESULTS: Graft and aortic ECs in primary culture did not produce significantly different amounts of PDGF in 72 hours, averaging 368 +/- 160 and 340 +/- 81 pg/microgram DNA, respectively. Graft SMCs in primary culture produced significantly more PDGF than aortic SMCs (584 +/- 343 and 113 +/- 94 pg/microgram DNA, respectively; p < 0.01). Graft SMC PDGF secretion remained greater than aortic SMC PDGF secretion through at least six cell passages. PDGF A-chain mRNA levels were not significantly different for aortic or graft ECs. The PDGF A-chain mRNA level was significantly higher for graft SMCs than aortic SMCs (2.44 +/- 0.67 and 1.45 +/- 0.57 pg/microgram, respectively; p < 0.03). Immunocytochemical analysis detected PDGF A-chain and B-chain protein in the ECs from both native aorta and graft as well as the subendothelial SMCs in the graft, but not in the SMCs of the native aorta. CONCLUSIONS: These results suggest that graft SMCs are functionally altered, producing more PDGF than aortic SMCs. PDGF produced by graft SMCs may contribute to the development of intimal hyperplasia.

Role of lysophosphatidylcholine in the inhibition of endothelial cell motility by oxidized low density lipoprotein.

Endothelial cell (EC) movement is required for the development and repair of blood vessels. We have previously shown that LDL oxidized by transition metals almost completely suppressed the wound-healing migratory response of vascular EC in vitro. We now report that lysophosphatidylcholine (lysoPC), a lipid component of oxidized LDL, has an important role in the antimigratory activity of the lipoprotein. Purified 1-palmitoyl lysoPC inhibited movement with a half-maximal activity at 12-15 micrometers, and near complete inhibition at 20 micrometers; the inhibitory concentration of lysoPC was consistent with its abundance in oxidized LDL. The inhibition was not due to cytotoxicity since protein synthesis was unaffected and since EC movement was restored after removal of lysoPC. Lysophospholipid activity was dependent on lipid structure. LysoPC's containing 1-position C16 or C18 saturated fatty acids were antimigratory, but those containing C < or = 14 saturated fatty acids or polyunsaturated fatty acids were not. The activity of 1-palmitoyl lysolipids with various head groups was examined. Lysophosphatidylinositol was more antimigratory than lysophosphatidylglycerol and lysophosphatidylcholine, which were more potent than lysophosphatidylserine and lysophosphatidylethanolamine. Monoglyceride was inactive while lysophosphatidate had promigratory activity. These results are consistent with head group size rather than charge as a critical determinant of activity. To show that lysophospholipids within an intact lipoprotein were active, LDL was treated with bee venom phospholipase A2 (PLA2). The modified lipoprotein inhibited EC movement to the same extent as iron-oxidized LDL and antimigratory activity correlated with the amount of lysoPC formed. To determine antimigratory activity of lysoPC present in oxidized LDL, lipid extracts from oxidized LDL were fractionated by normal phase HPLC. The fraction comigrating with lysoPC had nearly the same activity as the total extract confirming that lysoPC (or a co-eluting lipid) was a major antimigratory molecule in oxidized LDL. These studies demonstrate that lysoPC in oxidized LDL limit EC wound healing responses in vitro, and suggest a possible role for lysolipids in limiting endothelial regeneration after a denuding injury in vivo.

Ceruloplasmin enhances smooth muscle cell- and endothelial cell-mediated low density lipoprotein oxidation by a superoxide-dependent mechanism.

Cultured vascular smooth muscle cells (SMC) and endothelial cells (EC) stimulate low density lipoprotein (LDL) oxidation by free radical-mediated, transition metal-dependent mechanisms. The physiological source(s) of metal ions is not known; however, purified ceruloplasmin, a plasma protein containing 7 coppers, oxidizes LDL in vitro. We now show that ceruloplasmin also increases LDL oxidation by vascular cells. In metal ion-free medium, human ceruloplasmin increased bovine aortic SMC- and EC-mediated LDL oxidation by up to 30- and 15-fold, respectively. The maximal response was at 100-300 microg ceruloplasmin/ml, a level at or below the unevoked physiological plasma concentration. Oxidant activity was dependent on protein structure as a specific proteolytic cleavage or removal of one of the seven ceruloplasmin copper atoms inhibited activity. Three lines of evidence indicated a critical role for cellular superoxide (O2.) in ceruloplasmin-stimulated oxidation. First, the rate of production of O2. by cells correlated with their rates of LDL oxidation. Second, superoxide dismutase effectively blocked ceruloplasmin-stimulated oxidation by both cell types. Finally, O2. production by SMC quantitatively accounted for the observed rate of LDL oxidation. To show this, the course of O2. production by SMC was simulated by repeated addition of xanthine and xanthine oxidase to culture medium under cell-free conditions. Neither ceruloplasmin nor O2. alone increased LDL oxidation, but together they completely reconstituted the oxidation rate of ceruloplasmin-stimulated SMC. These results are the first to show that ceruloplasmin stimulates EC- and SMC-mediated oxidation of LDL and that cell-derived O2. accounts quantitatively for metal-dependent, free radical-initiated oxidation of LDL by these cells.

Inhibition of smooth muscle cell proliferation and migration in vitro by antisense oligonucleotide to c-myb.

PURPOSE: Smooth muscle cell (SMC) migration and proliferation are prominent features of intimal hyperplasia. Previous studies have shown that inhibition of c-myb inhibits arterial SMC proliferation. Our goal was to evaluate the effect of an antisense oligonucleotide targeted to c-myb on the proliferation and migration of SMC explanted from synthetic vascular grafts. METHODS: SMCs were enzymatically removed from aortas and Dacron grafts explanted from dogs (n = 5). For proliferation studies, quiescent SMCs were incubated with either 0.0, 0.5, 5.0, or 10.0 microM antisense (GTGTCGGGGTCTCCGGGC) or sense (GCCCGGAGACCCCGACAC) oligonucleotides to c-myb. Proliferation was measured after 24 hours by incorporation of [3H]thymidine. Migration was assessed 24 hours after a razor injury. RESULTS: Antisense to c-myb consistently inhibited proliferation and migration of both native aortic and graft SMCs in a dose-dependent fashion. At a concentration of 10 microM antisense oligonucleotide, aortic and graft SMC proliferation rates were 32% +/- 20% and 56% +/- 9% of control samples, respectively. At 25 microM antisense, the number of migrating aortic and graft SMCs decreased to 41.9% +/- 26.8% and 51.9% +/- 34.1% of control samples, respectively. CONCLUSIONS: Our results suggest that antisense oligonucleotides to c-myb may be useful in the inhibition of SMC proliferation and migration associated with development of intimal hyperplasia.