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T Maciag

Publications and source records attributed to T Maciag.

At least 91 records · Page 5Linked to original sources
PubMed1990

Structure-function relationships in sheep, mouse, and human prostaglandin endoperoxide G/H synthases.

Our studies are designed to determine which amino acid residues are involved in catalyzing the cyclooxygenase and hydroperoxidase activities of prostaglandin endoperoxide (PGG/H) synthase. We have deduced from complementary (c)DNAs the amino acid sequences of the sheep and mouse PGG/H synthases, and a portion of the human PGG/H synthase. These enzymes have amino acid sequences which are about 90% identical. Sequence similarities with putative heme binding regions of myeloperoxidase and thyroid peroxidase suggest that the sequence TI(L)WLREHNRV of PGG/H synthase contains the histidine (His309) which is the proximal heme ligand; the distal heme ligand may be His226 which is found in the sequence 222-KALGH-226. Using site-directed mutagenesis, we have replaced Ser530, the serine residue which is acetylated by aspirin, with Ala530 and with Asn530; the Ala530 mutant has both cyclooxygenase and hydroperoxidase activity, while the Asn530 mutant lacks cyclooxygenase activity but retains hydroperoxidase activity. These results establish that the hydroxyl group of Ser530 is not essential for catalysis or substrate binding and suggest that a bulky group at position 530, such as that introduced by aspirin acetylation, prevents arachidonate binding to the cyclooxygenase active site. Finally, we have found that tetranitromethane causes irreversible inactivation of cyclooxygenase activity and that the enzyme is protected from inactivation when ibuprofen is included in the reaction mixture. These results suggest that there is an essential tyrosine at the active site of PGG/H synthase.

Amino Acid Sequence↗
PubMed1989

Interleukin 1 regulates synthesis of amyloid beta-protein precursor mRNA in human endothelial cells.

We have analyzed the modulation of amyloid beta-protein precursor (APP) gene expression in human umbilical vein endothelial cells (HUVEC). The level of the APP mRNA transcripts increased as HUVEC reached confluency. In confluent culture the half-life of the APP mRNA was 4 hr. Treatment of the cells with human-recombinant interleukin 1 (IL-1), phorbol 12-myristate 13-acetate, or heparin-binding growth factor 1 enhanced the expression of APP gene in these cells, but calcium ionophore A23187 and dexamethasone did not. The protein kinase C inhibitor 1-(isoquinolinsulfonyl)-2-methylpiperazine (H7) inhibited IL-1-mediated increase of the level of APP transcripts. To map IL-1-responsive elements of the APP promoter, truncated portions of the APP promoter were fused to the human growth hormone reporter gene. The recombinant plasmids were transfected into mouse neuroblastoma cells, and the cell medium was assayed for the human growth hormone. A 180-base-pair region of the APP promoter located between position -485 and -305 upstream from the transcription start site was necessary for IL-1-mediated induction of the reporter gene. This region contains the upstream transcription factor AP-1 binding site. These results suggest that IL-1 upregulates APP gene expression in HUVEC through a pathway mediated by protein kinase C, utilizing the upstream AP-1 binding site of the APP promoter.

Amyloid↗
PubMed1989

Heparin-binding growth factor 1 induces the formation of organoid neovascular structures in vivo.

One of the promises of modern molecular biology has been the opportunity to use genetically modified human cells in a patient to permanently restore inborn errors of metabolism. Although it has been possible to introduce genes into mammalian cells and to control their expression, it has proven difficult to introduce mammalian cells as carriers of the modified genetic information into hosts. The successful implantation of selective cells cannot be achieved without adequate vascular support, an essential step toward integration and reconstitution of a new biological function. Although a partial solution to this problem has been found by inducing specific site-directed neovessel formation using heparin-binding growth factor 1 (HBGF-1) adsorbed to a collagen matrix, these implants function for only a short period (weeks). We now report the formation of organoid neovascular structures using polytetrafluoroethylene fibers coated with collagen and HBGF-1 implanted in the peritoneal cavity of the rat. The organoid structures contained readily visible vascular lumina and nonvascular structures that resemble nerve tissue. It was also possible to demonstrate that the vascular system on the implant is continuous with the vascular tree of the host. This feature was used to demonstrate that the organoid structures are capable of sustaining the biological function of implanted normal rat hepatocytes over long periods of time (months) in the homozygous Gunn rat, thereby facilitating future applications involving the delivery of new genetic information.

Animals↗
PubMed1989

Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.

Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.

Animals↗
PubMed1989

Pharmacokinetics and distribution of heparin-binding growth factor I (endothelial cell growth factor) in the rat.

Heparin-binding growth factor I (HBGF I), previously designated as endothelial cell growth factor, is a potent mitogen for endothelial cells in vitro, which may prove useful for promoting endothelial regeneration in vivo. Analysis of the pharmacokinetics and organ distribution of HBGF I is necessary before use of HBGF I as a pharmacological agent. Consequently, pharmacological studies were carried out with [125I]HBGF I in the rat. Intravenous injections of HBGF I were given with or without heparin (2.5 units/ng HBGF I). Blood concentrations of HBGF I decreased by one half 17 seconds after HBGF I bolus. This time was prolonged to 60 seconds when HBGF I was injected with heparin. The elimination half-life of HBGF I was 14 minutes in the presence of heparin. The highest concentrations of HBGF I following intravenous bolus were found in kidney, liver, and spleen, and the lowest in fat and brain. Heparin increased HBGF I concentrations in blood and all organs measured except kidney, which was significantly decreased (p less than 0.01). Intact HBGF I was recoverable from blood 5 minutes following intravenous administration. HBGF I underwent near-complete proteolytic digestion after more prolonged ex vivo incubation with rat plasma, but HBGF I was protected from proteolysis when incubations were conducted in the presence of heparin. Thus, it is feasible that HBGF I can be administered as a pharmacological agent in the presence of heparin. Further studies assessing acceleration of in vivo endothelial growth using HBGF I with heparin appear warranted.

Animals↗
PubMed1988

Site-directed neovessel formation in vivo.

Angiogenesis is an important component of organogenesis and wound repair and occurs during the pathology of oncogenesis, atherogenesis, and other disease processes. Thus, it is important to understand the physiological mechanisms that control neovascularization, especially with methods that permit the molecular dissection of the phenomenon in vivo. Heparin-binding growth factor-1 was shown to bind to collagen type I and type IV. When complexed with gelatin, heparin-binding growth factor-1 can induce neovascularization at polypeptide concentrations that are consistent with the biological activity of the mitogen in vitro. The adsorption strategy induces rapid blood vessel formation at and between organ- and tissue-specific sites and permits recovery of the site-specific implant for examination and manipulation by molecular methods.

Animals↗
PubMed1988

Regulation of plasminogen activator inhibitor-1 mRNA in human endothelial cells.

The plasminogen activator inhibitor (PAI-1) from endothelial cells is a potentially important regulator of plasminogen activator activity. Cultured human endothelial cells increase their PAI-1 production upon stimulation with LPS and TNF, agents that are known to cause an increase in PAI-1 levels in vivo. We isolated a PAI-1 cDNA probe, and by RNA hybridization analysis studied the regulation of PAI-1 mRNA synthesis in human endothelial artery cells. Freshly isolated endothelial cells do not contain detectable amounts of PAI-1 mRNA, but after adherence and incubation for 18 h in growth medium produce considerable amounts of PAI-1 activity and contain PAI-1 mRNA levels comparable to those found in subcultured cells. When subcultured endothelial cells are incubated for 6 h with LPS or TNF, both species of PAI-1 mRNA increase 10 to 20 fold, while PAI-1 activity in the growth medium increases only 1.5 to 2 fold. Stimulation of endothelial cells in the presence of cycloheximide (CHX) results in superinduction of mainly the 3.0 kb PAI-1 mRNA. The 3' end of this mRNA contains a 60 bp AT-rich sequence, that resembles 3' sequences present in a number of other genes superinducible with CHX.

Base Sequence↗
PubMed1988

Heparin protects heparin-binding growth factor-I from proteolytic inactivation in vitro.

Heparin inhibits proteolytic digestion of heparin-binding growth factor-I (HBGF-I) by trypsin, plasmin and other proteases. This property is lost after thermal denaturation of HBGF-I, suggesting that a heparin:HBGF-I structural interaction rather than a heparin:trypsin interaction is responsible for the resistance of HBGF-I to digestion with trypsin. Heparin is also able to partially protect HBGF-I from thermal denaturation as demonstrated by the ability of heparin to protect HBGF-I from trypsin digestion. The protective effect of heparin is dependent upon the concentration of heparin as well as temperature and duration of denaturation. Autoradiography of 125I-HBGF-I incubated with human umbilical vein endothelial cells demonstrates near complete protection of HBGF-I from proteolytic modification when the incubation is performed in the presence of heparin. These data suggest that (i) the mechanism of the heparin-induced increase in human endothelial cell number at confluence involves the protection of HBGF-I by heparin against proteolytic inactivation and (ii) heparin provides conformational stability to the proteolytic growth factor which reduces the susceptibility of HBGF-I to denaturation.

Animals↗
PubMed1988

Internalization and degradation of heparin binding growth factor-I by endothelial cells.

The fate of 125I-labeled heparin binding growth factor I (125I-HBGF-I) after binding to its cell surface receptor has been studied using murine lung capillary endothelial cells (LEII). Binding of 125I-HBGF-I to its receptor at 4 degrees C shows pH dependence with optimal binding at pH 6.5-7.5. The majority (approximately 80%) of 125I-HBGF-I bound to cells at 4 degrees C can be removed by washing with low pH medium, but rapidly becomes acid resistant upon shifting cells to 37 degrees C, with 50% of the 125I-HBGF-I becoming acid resistant after 20 minutes. Electrophoretic analysis of internalized 125I-HBGF-I shows that degradation begins approximately 2 hours after internalization with the appearance of two major labeled fragments of Mr 15,000 and Mr 10,000. Degradation of internalized 125I-HBGF-I is inhibited by the lysosomotropic agent chloroquine. These data suggest that cell-associated 125I-HBGF-I is rapidly internalized and directed to a lysosomal cellular compartment where it is slowly degraded.

Animals↗
PubMed1988

Regulation of neuroblast proliferation by hormones and growth factors in chemically defined medium.

Pure neuronal cultures prepared from 6-day-old embryonic chick brains incorporated [3H]-thymidine in serum-free medium up to the 4th day in culture. The addition of insulin any time within this culture period caused an increase in thymidine incorporation. This increase in [3H]-thymidine was correlated with an increase in cell number and percentage of labeling index. Triiodothyronine and endothelial cell growth factor were also active in stimulating [3H]-thymidine incorporation into chick neuroblasts. The effect of these trophic agents is unique since a variety of known mitogens tested were negative.

Animals↗
PubMed1988

Heparin-binding growth factor-I (endothelial cell growth factor) binds to endothelium in vivo.

Heparin-binding growth factor-I (HBGF-I) produces a significant increase in endothelial cell replication in vitro and may prove useful for endothelial regeneration and endothelial seeding in vivo. Heparin enhances the mitogenic effects of HBGF-I in vitro and may be an important adjunct to its pharmacologic use. Binding studies in the rat were undertaken to determine the feasibility of use of HBGF-I in vivo. For saturation studies, 125I-labeled HBGF-I was administered intravenously at various concentrations with heparin (approximately 5 U/ml blood volume). Further binding studies were conducted with the use of a constant concentration of 125I-labeled HBGF-I (40 ng) with or without heparin (2.5 U/ng HBGF-I). In each case, the rat was perfused with ice-cold saline solution 5 minutes after drug administration and the aorta or carotid arteries were harvested. 125I-labeled HBGF-I with heparin demonstrated saturation binding to rat aortic endothelium at an approximate blood concentration of 10 ng/ml. Scatchard plot analysis of in vivo data revealed a binding constant (KD) of 1.60 +/- 0.004 x 10(-9) mol/L compared with 2 to 8 x 10(-10) mol/L obtained for in vitro binding. Receptor number per cell was approximately 3000 for rat aortic endothelium, compared with a receptor number of 2000 to 20,000 for several endothelial cell lines, including bovine aortic endothelial cells, determined in vitro. 125I-labeled HBGF-I binding to uninjured rat carotid endothelium was significantly increased (p = 0.0001) by heparin (463 +/- 302 cpm to 1172 +/- 403 cpm).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

An angiogenic growth factor is expressed in human glioma cells.

Progression to increased malignancy frequently occurs in human brain tumors of glial origin and usually involves neovascularization--a massive proliferation of endothelial cells into the tumor tissue. We have shown previously that subversion of a normal growth factor-related pathway is frequently associated with human gliomas. Here we show that human glioma cell lines express the gene encoding the angiogenic peptide endothelial cell growth factor (ECGF) or acidic fibroblast growth factor (a-FGF) and that an ECGF-like polypeptide is produced by these cells. The glioma-derived growth factor was partially purified from cell extracts by heparin-Sepharose affinity chromatography where it eluted at 1.5 M sodium chloride. On reversed-phase h.p.l.c., growth factor activity for endothelial cells was eluted at the same concentration of acetonitrile as found for bovine brain-ECGF, also a potent mitogen for endothelial cells. Moreover, human glioma cells possess specific cell surface receptors for ECGF and are mitogenically stimulated by exogenous addition of this growth factor. Glioma derived-ECGF may therefore have a dual influence: first, by autocrine growth-stimulation of human gliomas and, second, by paracrine-stimulation of endothelial cell proliferation which results in neovascularization of the tumor tissue.

Cell Line↗
PubMed1987

Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor).

The control of vascular endothelial and smooth muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical vein endothelial cells, express HBGF-I mRNA. Smooth muscle cells also synthesize an HBGF-I-like polypeptide since (i) extract prepared from smooth muscle cells will compete with 125I-labeled HBGF-I for binding to the HBGF-I cell surface receptor, and (ii) the competing ligand is eluted from heparin-Sepharose affinity resin at a NaCl concentration similar to that required by purified bovine brain HBGF-I and stimulates endothelial cell proliferation in vitro. Furthermore, like endothelial cells, smooth muscle cells possess cell-surface-associated HBGF-I receptors and respond to HBGF-I as a mitogen. These results indicate the potential for an additional autocrine component of vascular smooth muscle cell growth control and establish a vessel wall source of HBGF-I for endothelial cell division in vivo.

Animals↗
PubMed1987

Inhibition of endothelial cell proliferation by gamma-interferon.

Endothelial cell growth factor (ECGF) is a potent polypeptide mitogen for endothelial cells and fibroblasts. The mitogenic effects of ECGF are inhibited by the lymphokine gamma-interferon (gamma-IFN) in a dose-dependent manner. Gamma-IFN also induces a unique change in endothelial cell morphology which is maximally expressed in the presence of ECGF. The antiproliferative and phenotypic modulatory effects of gamma-IFN on endothelial cells are reversible. Inhibition of ECGF-induced endothelial cell proliferation by gamma-IFN is accompanied by a concentration- and time-dependent decrease in binding of 125I-ECGF to the endothelial cell surface. Scatchard analyses of the binding data in the presence and absence of gamma-IFN demonstrate a decrease in the number of ECGF-binding sites rather than a decrease in ligand affinity for the receptor. Cross-linking experiments with disuccinimidyl suberate demonstrate a decrease in the 170,000 Mr cross-linked receptor-ligand complex. These data suggest that gamma-IFN inhibits endothelial cell proliferation by a mechanism which involves growth factor receptor modulation.

Cell Division↗
PubMed1987

Effects of endothelial cell growth factor on bone remodelling in vitro.

Endothelial cell growth factor (ECGF) alpha was studied for its effects on bone formation in cultured fetal rat calvariae and on bone resorption in cultured fetal rat long bones. ECGF at 0.1-100 ng/ml stimulated [3H]thymidine incorporation into DNA, an effect enhanced by heparin. Treatment with ECGF for 24 h decreased the incorporation of [3H]proline into collagen but treatment for 48-96 h increased collagen and noncollagen protein synthesis, an effect that was concomitant with an increase in DNA content. ECGF did not alter collagen degradation in calvariae or 45Ca release from long bones, which indicated it had no effect on bone resorption. Although ECGF increased prostaglandin E2 concentrations, its effect on DNA synthesis was not prostaglandin-mediated. In conclusion, ECGF stimulates calvarial DNA synthesis, which is an effect that results in a generalized increase in protein synthesis, but ECGF has no effect on matrix degradation or bone resorption.

Animals↗
PubMed1986

Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization.

Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.

Base Sequence↗
PubMed1986

The characterization of the receptor for endothelial cell growth factor by covalent ligand attachment.

Cellular receptors for endothelial cell growth factor (ECGF) have been demonstrated on several cell types by binding of 125I-ECGF in a specific and saturable manner (Schreiber, A. B., Kennedy, J., Kowalski, J., Friesel, R., Mehlman, T., and Maciag, T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6138-6142). Here we report the covalent cross-linking of 125I-ECGF to a polypeptide present on the surface of the plasma membrane of murine lung capillary endothelial cells by the homobifunctional reagent, disuccinimidyl suberate. Cross-linking of cell surface associated 125I-ECGF yields a major polypeptide with an apparent molecular weight of 150,000. Experiments demonstrated that the cross-linked polypeptide complex represents 125I-ECGF covalently bound specifically to a cell surface receptor because: covalent modification of the polypeptide was inhibited by excess, unlabeled ECGF; preincubation of cells with unlabeled ECGF at 37 degrees C significantly reduced cross-linking while incubation at 4 degrees C did not; other polypeptide growth factors do not compete with 125I-ECGF for cross-linking to the ECGF receptor; labeling of the polypeptide did not take place in the absence of DSS; and cells previously shown to have a paucity of ECGF receptors did not yield a cross-linked labeled receptor. These data suggest that the mitogenic events mediated by ECGF occur after occupancy of the specific cell surface polypeptide and suggest that these events are relevant to ECGF-induced signal transduction across the endothelial cell plasma membrane.

Animals↗