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T Maciag

Publications and source records attributed to T Maciag.

At least 109 records · Page 6Linked to original sources
PubMed1986

Structural evidence that endothelial cell growth factor beta is the precursor of both endothelial cell growth factor alpha and acidic fibroblast growth factor.

Two endothelial cell growth factors (ECGF) have been purified from bovine brain and termed alpha- and beta-ECGF [Burgess, W. H., Mehlman, T., Friesel, R., Johnson, W. V. & Maciag, T. (1985) J. Biol. Chem. 260, 11389-11392]. Amino acid sequence analysis indicates that beta-ECGF represents a 20 amino acid amino-terminal extension of alpha-ECGF and a 14 amino acid amino-terminal extension of acidic fibroblast growth factor. These data indicate that both alpha-ECGF and acidic fibroblast growth factor may be derived from beta-ECGF by posttranslational processing. Analysis of the amino-terminal 14 residues of beta-ECGF by fast-atom-bombardment mass spectrometry established the amino acid sequence of this region and the identity of the blocking group at the amino terminus (acetyl).

Amino Acid Sequence↗
PubMed1986

Isolation and sequencing of a cDNA clone homologous to the v-sis oncogene from human endothelial cells.

A clone containing the 3' end of the mRNA for the human c-sis gene (homologous to the B chain of platelet-derived growth factor) was isolated from a cDNA library derived from human umbilical vein endothelial cells and then sequenced. The analysis of possible translation products in all three reading frames indicated that the A chain of platelet-derived growth factor was not coded for within the 3' end of the c-sis mRNA. The 3' end of the mRNA for c-sis is contained in or adjacent to exon 6.

Base Sequence↗
PubMed1986

Propagation and morphologic phenotypes of human umbilical cord artery endothelial cells.

Human umbilical cord artery endothelial cells can be propagated on fibronectin-coated dishes for approximately 50 cumulative population doublings in the presence of crude preparations of endothelial cell growth factor (ECGF) and serum. The cells were characterized by immunofluorescent staining and their ultrastructure. Different morphologic phenotypes could be demonstrated: closely attached cell monolayers, atypical cells, giant cells, tube-like structures. The formation of tube-like structures can be induced by proteolytic modification of fibronectin. Our data demonstrate that umbilical arteries may provide an excellent source for the routine serial cultivation of human arterial endothelial cells.

Cell Division↗
PubMed1985

Multiple forms of endothelial cell growth factor. Rapid isolation and biological and chemical characterization.

Endothelial cell growth factor (ECGF) can be rapidly purified from bovine brain to high specific activity using heparin-Sepharose affinity chromatography. Purification of the mitogen by this method results in relatively high yields of the polypeptide (10 to 100 micrograms/kg of tissue) with biological activity on murine and human endothelial cells in the picogram range. The product obtained is a mixture of two single-chain polypeptides with apparent molecular weights of 17,000 (alpha-ECGF) and 20,000 (beta-ECGF) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two forms of ECGF can be separated by either NaCl gradient elution from heparin-Sepharose or reversed-phase high pressure liquid chromatography. The two polypeptides are related on the basis of similar: amino acid compositions, affinity for heparin-Sepharose, cyanogen bromide and trypsin-derived cleavage products, and biological activity. Furthermore, the cyanogen bromide fragments derived from the two forms of ECGF also possess similar amino acid compositions and mobilities on sodium dodecyl sulfate gels. These data suggest that there are at least two discrete molecular forms of ECGF in bovine brain and that these two molecules are structurally related.

Amino Acids↗
PubMed1985

Modulation of the sis gene transcript during endothelial cell differentiation in vitro.

Endothelial cells, which line the interior walls of blood vessels, proliferate at the site of blood vessel injury. Knowledge of the factors that control the proliferation of these cells would help elucidate the role of endothelial cells in wound healing, tumor growth, and arteriosclerosis. In vitro, endothelial cells organize into viable, three-dimensional tubular structures in environments that limit cell proliferation. The process of endothelial cell organization was found to result in decreased levels of the sis messenger RNA transcript and increased levels of the messenger RNA transcript for fibronectin. This situation was reversed on transition from the organized structure to a proliferative monolayer. These results suggest a reciprocity for two biological response modifiers involved in the regulation of endothelial cell proliferation and differentiation in vitro.

Cell Differentiation↗
PubMed1985

The stimulation of normal human melanocyte proliferation in vitro by melanocyte growth factor from bovine brain.

Cell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10(-9) M), hydrocortisone, (5 X 10(-5) M), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10(-10) M), and bovine brain extract (150 micrograms/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor-supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]-Dopa incorporation. The growth factor-supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline-labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co-cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.

Animals↗
PubMed1985

Interaction of endothelial cell growth factor with heparin: characterization by receptor and antibody recognition.

Endothelial cell growth factor (ECGF) binds specifically in vitro to membrane receptors present on the surface of several cell types, including murine and human endothelial cells and fibroblasts. Monoclonal antibodies prepared against ECGF that inhibit the mitogenic activity of the growth factor prevent receptor occupancy by the ligand. Heparin interacts structurally with ECGF [Maciag, T., Mehlman, T., Friesel, R. & Schreiber, A. B. (1984) Science 225, 932-935], potentiates the mitogenic activity of the polypeptide, restores the biological activity to inactivate ECGF, enhances the affinity of the ligand to cell surface receptors, and modifies antibody recognition of ECGF. These data suggest that the association between heparin and ECGF induces a conformational change in the polypeptide that increases or stabilizes the biological activity of the mitogen.

Cell Division↗
PubMed1985

A unique family of endothelial cell polypeptide mitogens: the antigenic and receptor cross-reactivity of bovine endothelial cell growth factor, brain-derived acidic fibroblast growth factor, and eye-derived growth factor-II.

Bovine brain, hypothalamus, pituitary, and retina contain potent anionic polypeptide mitogens for endothelial cells. Immunological assays using murine monoclonal antibodies against bovine endothelial cell growth factor (ECGF) and radioreceptor assays using [125I]ECGF were performed to determine the cross-reactivity of ECGF with bovine acidic pI brain-derived fibroblast growth factor (acidic FGF) and bovine eye-derived growth factor-II [EDGF-II). We observed that acidic FGF and EDGF-II are recognized by anti-ECGF monoclonal antibodies and compete with [125I] ECGF for receptor occupancy. Furthermore, the biological activity of ECGF, acidic FGF, and EDGF-II is potentiated by the glycosaminoglycan, heparin. These results argue that ECGF, acidic FGF, and EDGF-II belong to a common family of polypeptide growth factors.

Animals↗
PubMed1985

Human endothelial cells are chemotactic to endothelial cell growth factor and heparin.

The response of human endothelial cell migration to various extracellular matrix components and growth factors has been assessed. Human endothelial cells demonstrate increased chemotaxis and chemokinesis when placed in a modified Boyden chamber with endothelial cell growth factor (ECGF) used at a concentration of 10(-9) M. Anti-ECGF antibody inhibits the chemotactic response. Heparin (10(-8) to 10(-10) M) was also chemotactic and was shown to potentiate the chemotactic activity of ECGF. Although laminin, fibronectin, the polypeptide (epidermal, fibroblast, and nerve) growth factors, and collagen types I, II, III, IV, and V demonstrate a chemotactic response, these activities were one third to one half less than observed with ECGF. These data suggest that ECGF and heparin may play a significant role as response modifiers of human endothelial cell migration which may be relevant to tumor metastasis, wound healing, and atherogenesis.

Cells, Cultured↗
PubMed1984

Heparin binds endothelial cell growth factor, the principal endothelial cell mitogen in bovine brain.

Endothelial cell growth factor (ECGF), an anionic polypeptide mitogen, binds to immobilized heparin. The interaction between the acidic polypeptide and the anionic carbohydrate suggests a mechanism that is independent of ion exchange. Monoclonal antibodies to purified bovine ECGF inhibited the biological activity of ECGF in crude preparations of bovine brain. These data indicate that ECGF is the principal mitogen for endothelial cells from bovine brain, that heparin affinity chromatography may be used to purify and concentrate ECGF, and that the affinity of ECGF for heparin may have structural and perhaps biological significance.

Animals↗
PubMed1984

Characterization and partial purification of keratinocyte growth factor from the hypothalamus.

An extract of bovine hypothalamus is known to be mitogenic for human keratinocytes in vitro. In order to identify the responsible substance(s), biochemical characterization and subsequent bioassay of the extract in a serum-free culture system were performed. The keratinocyte growth-promoting activity of the hypothalamic extract was unaffected by heating (100 degrees C, 10 min); acidification to pH 3.3; or by exposure to lipase, RNAase, or proteolytic enzymes; but was abolished by alkalinization to pH 11. An approximate molecular weight of 1,700 daltons was determined by elution on a calibrated Sephadex G-25 column, and an approximate pl of 3.5 was determined by isoelectric focusing. Optimal concentrations of the crude extract (150-300 micrograms/ml) increased keratinocyte growth approximately 50-fold compared to control cultures lacking the extract. Partial purification resulted in a preparation biologically active at 30 ng/ml protein equivalent and was consistent with the presence of a single mitogen which we have termed keratinocyte growth factor (KGF). Mitogenic activity for human melanocytes, dermal fibroblasts, and endothelial cells, present in the crude hypothalamic extract, was lacking in heat-treated preparations that contained KGF. Optimal concentrations of purified epidermal growth factor and ethanolamine, the only remotely similar substances previously reported to augment keratinocyte growth in vitro, could not substitute for KGF in the serum-free culture system. Keratinocyte growth-promoting activity comparable to that observed in bovine hypothalamic extracts was present in human hypothalamic extracts prepared in the same manner.

Animals↗
PubMed1984

Angiogenesis.

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Angiogenesis Inducing Agents↗
PubMed1983

Autocrine and paracrine growth stimulation of cells derived from human skin.

A sensitive serum-free culture system was used to demonstrate that cells derived from normal human skin release soluble mediators which can modulate keratinocyte, melanocyte, and fibroblast growth in vitro. In M199 supplemented with epidermal growth factor, hydrocortisone, insulin, transferrin, triidothyronine, bovine serum albumin, and an extract of bovine hypothalamus, keratinocytes underwent approximately three to five cumulative population doublings (CPD) over a 7-day period. Addition of 20% keratinocyte-conditioned medium more than doubled keratinocyte growth and increased fibroblast growth 25-40% above controls. Dermal fibroblasts maintained in the same serum-free hormone-supplemented medium (SFHSM) alone underwent approximately 4.5-5.5 CPD over a 7-day period; 20% fibroblast-conditioned medium increased fibroblast growth 50-80% and increased keratinocyte growth 30-50%. Melanocytes maintained in the same SFHSM underwent approximately 1 CPD over a 14-day period and 2 CPD if the medium was supplemented with 2% fetal bovine serum (FBS). Addition of 20% melanocyte-conditioned medium more than doubled melanocyte growth in either SFHSM or in medium containing 2% FBS, but decreased both keratinocyte and fibroblast growth up to 30-60%. None of the conditioned media altered cellular morphology. These data provide the first demonstration of mutual growth modulation by cell types normally contiguous in vivo and expand existing evidence for autocrine and paracrine growth regulation by normal human cells.

Adult↗
PubMed1982

High and low molecular weight forms of endothelial cell growth factor.

Endothelial cell growth factor (ECGF) is a neural-derived mitogen capable of stimulating the proliferation of quiescent populations of human umbilical vein endothelial cells (HUV-EC). ECGF has been isolated from bovine hypothalamus in two forms: a high molecular weight from (high Mr ECGF, greater than 70,000) and a low molecular weight form form (low Mr ECGF, 17,000 to 25,000). Isoelectric focusing of high Mr ECGF and low Mr ECGF revealed that both Mr forms possess similar anionic isoelectric points (pI 4 to 6). High Mr ECGF can be converted into low Mr ECGF by mild acidification of the high Mr ECGF complex. Low Mr ECGF prepared by the high Mr to low Mr transition procedure is biologically active at 100 ng/ml in the low density HUV-EC growth assay and is capable of stimulating DNA synthesis in Balb/c 3T3 cells at 10 ng/ml. The two Mr forms of ECGF are related since they (i) share similar biological activity, (ii) possess similar isoelectric points, and (iii) high Mr ECGF can be converted into low Mr ECGF by mild treatment with acetic acid. Furthermore, the high Mr and low Mr forms of ECGF are not related to the cationic bovine brain fibroblast growth factor.

Animals↗
PubMed1982

Growth of human foreskin fibroblasts in a serum-free, defined medium without platelet-derived growth factor.

The hormones which support growth, in vitro, of normal, neonatal human foreskin fibroblasts were determined. Whereas thrombin and hydrocortisone were major growth stimulants, platelet-derived growth factor was not. Human foreskin fibroblasts grew in a serum-free, biochemically defined medium consisting of epidermal growth factor (100 ng/ml), insulin (100 ng/ml), transferrin 10 micrograms/ml), thrombin (1 microgram/ml), ascorbic acid (10 micrograms/ml), and hydrocortisone (5 x 10(-5) M) in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12, supplemented with ovalbumin (1 mg/ml) and trace elements. The growth achieved was comparable to that achieved with 5% fetal bovine serum. Neither platelet-derived growth factor, fibroblast growth factor, nor somatomedin activity increased proliferation. This serum-free medium, designated Defined Medium F, provides a biochemically defined system for growth and limited subcultivation of human foreskin fibroblasts in vitro.

Blood Platelets↗
PubMed1982

Attachment and growth of human keratinocytes in a serum-free environment.

Using a serum-free system, we have investigated the influence of human fibronectin (HFN) and selected growth factors (GF) on the attachment and growth of normal human keratinocytes in vitro. Single-cell suspensions of keratinocytes from near-confluent primary plates, plated on 5-10 microgram/cm2 HFN, showed approximately 30-40% attachment after 2-24 hours of incubation at 37 degrees C, compared with 4-6% attachment on uncoated platic plates. Percentage of attached cells was independent of seed density, tissue donor age, in vitro culture age, or medium composition, while subsequent cellular proliferation was strongly dependent on these factors. Keratinocytes grown on an adequate HFN matrix in a previously described hormone-supplemented medium (Maciag et al., 1981a) achieved four to eight population doubling over 7-12 days at densities greater than or equal to 104 cell/cm2. Removal of most GF individually from the medium had little or no effect on growth, while removal of epidermal growth factor (EGF) alone reduced growth by 30-35% and removal of bovine brain extract (BE) alone reduced growth by approximately 90%. Conversely, EGF alone in basal medium supported approximately 10% control growth, BE alone supported 30-40% control growth, and the combination of EGF and BE approximately 70%. In addition to its major effect on proliferation in this system, BE was necessary to preserve normal keratinocyte morphology and protein production. These findings expand earlier observations that HFN facilitates keratinocyte attachment in vitro and that a brain-derived extract can exert a major positive influence on cultured keratinocytes.

Cell Adhesion↗
PubMed1982

Vascular smooth muscle cell growth kinetics in vivo in aged rats.

Age is a risk factor in the development of atherosclerosis. In this study we investigated the hypothesis that proliferation of vascular smooth muscle cells (SMCs), an integral part of atherosclerotic plaque formation, changes with age. SMC growth kinetics of old rats (21-24 months) were compared to those of young adult rats (3-4 months). Rat aortas were denuded of their endothelium and the animals were killed after [3H]thymidine and Evans blue injections at 0-28 days after denudation. Incorporation of [3H]thymidine into SMC peaked in the young animals by day 2, whereas the older animals responded to endothelial removal with greater incorporation at day 2 and a more sustained rate of incorporation peaking at day 4. The [3H]thymidine incorporation curves decreased sharply from their peaks at 2 and 4 days, respectively, and paralleled each other after day 7. [3H]Thymidine uptake reflected the subsequent SMC intimal growth as measured morphometrically, with old animals showing greater numbers of intimal SMC than did the younger animals. The difference in response of SMC to injury with age suggests that aging produces a change in the vascular SMC that enhances proliferation. This change in response implies that the more pronounced atherosclerotic plaque growth seen with aging may be a result of an age-related increase in response to injury rather than merely the accumulation of time-related intimal change.

Aging↗