Suppression of tumor-associated hyperfibrinogenemia and free fatty acidemia with p-phenoxybenzalbutyrate (clofibrate).

Previous work in our laboratory has indicated that free fatty acids stimulate synthesis of fibrinogen by the liver. The effect of the hypolipidemic agent clofibrate on hyperfibrinogenemia associated with tumors was evaluated by monitoring clofibrate-induced changes in plasma fibrinogen concentration and biosynthesis of the protein in Buffalo rats implanted with a localized, nonmetastasizing neoplasm derived from a tumorigenic hepatoma cell line (HTC4). In tumor-bearing animals not treated with clofibrate, cancer growth was associated with elevated rates of fibrinogen synthesis and a doubling of plasma fibrinogen concentrations. Plasma free fatty acid concentrations and serum free fatty acid/albumin molar ratios were also increased in tumor-bearing rats. Treatment with clofibrate in doses which normalized the plasma free fatty acid/albumin ratio also prevented the tumor-associated rise in plasma fibrinogen. Rates of fibrinogen synthesis were lowered significantly in clofibrate-treated animals. Tumor growth was not affected by clofibrate. These results indicate that hyperfibrinogenemia associated with nonmetastasizing tumors may reflect changes in lipid metabolism which are neutralized by clofibrate. Thus, treatment with clofibrate or other hypolipidemic agents should be evaluated in cancer patients with elevated plasma fibrinogen levels and their attendant complications.

Responses of rat and chick chondrocytes and rate hepatoma cells to plasma fractions with insulin-like and growth-promoting activities.

Plasma protein Cohn fraction IV-1 was extracted and fractionated according to the method of Van Wyk et al.[1], with the omission of the Sephadex G-75 step. The fractionated polypeptides were investigated for insulin-like activity (lipid synthesis and lactate production) and growth-stimulatory activity (DNA synthesis) in three bioassay systems: rat hepatoma cells, and rat and chick chondrocyte suspensions. Binding of each fraction to insulin receptors in isolated plasma membranes was also determined. Three peptide subfractions, with molecular weights ranging from 400 to 20 000, stimulated lipid synthesis, lactate production and DNA synthesis in all three bioassay systems. There were distinct species- and cell-type-specific variations in the quantitative patterns produced by each subfraction. In contrast, authentic insulin (0.005-0.1 ng/ml) had no effect on DNA and lipid synthesis anad lactate production in hepatoma cells and chondrocytes. One of the three active subfractions and two relatively inactive fractions displaced insulin from its receptors. These observations indicate that acid-ethanol extracts of plasma contain a variety of peptide factors with insulin-like and growth-promoting effects whose expression in different cell types is modulated by inherent properties of each cell type which are determined genetically presumably. Surface receptors for authentic insulin do not appear to play an essential role in the insulin-like activities of the isolated plasma fractions.

Surface analysis and depth profiles of calcium in hepatoma cells during pyruvate-induced DNA synthesis.

Induction of DNA synthesis is associated with increased uptake of calcium in cultured cells. Calcium distribution within the plasma membrane and adjacent cytoplasmic layers of hepatoma cells was investigated with X-ray photoelectron spectroscopy and oxygen plasma etching. Cells in minimal growth medium initiate active DNA synthesis 16 h after addition of sodium pyruvate. Cells stimulated with pyruvate and pyruvate-free controls were analysed by X-ray photoelectron spectroscopy--oxygen plasma etching at 0--40 A (layer I), 0--450 A (layer II) and 0--4000 A (layer III from the outer cell surface. Calcium concentrations were elevated in induced cells compared with controls: +20% in layer I, +60% in layer II, and +300% in layer III. As the plasma membrane is 90--120 A thick, these results indicate that pyruvate-induced DNA synthesis is preceded by an increase in calcium, most marked in the cytoplasm subjacent to the plasma membrane, moderate at its inner surface, and minimal at its outer surface.

Stimulation of collagen synthesis in fibroblast cultures by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+.

Glycyl-L-histidyl-L-lysine (GHK) is a tripeptide with affinity for copper(II) ions and was isolated from human plasma. This peptide appears to play a physiological role in wound healing. We report the stimulating effect of GHK-Cu on collagen synthesis by fibroblasts. The stimulation began between 10(-12) and 10(-11) M, maximized at 10(-9) M, and was independent of any change in cell number. The presence of a GHK triplet in the alpha 2(I) chain of type I collagen suggests that the tripeptide might be liberated by proteases at the site of a wound and exert in situ healing effects.

Increased ratio of plasma free fatty acids to albumin during normal aging and in patients with coronary heart disease.

The ratio of free fatty acids (FFA) to albumin, its carrier protein, was determined in 118 healthy men of ages 20 to 69 years and in 83 patients with coronary heart disease (CHD) of ages 33 to 69 years. During aging in normal men, this ratio increased progressively from an average value of 0.755 +/- 0.061 in the age 20-29 group to a value of 1.042 +/- 0.105 in the 60-69-year-old group. In patients with CHD this ratio was approximately 18% higher in each 10-year cohort than the corresponding control value, rising from a value of 1.029 +/- 0.081 in the 30-39-year-old group to a value of 1.212 +/- 0.106 in the 60-69-year-old group. The compositional spectrum of FFA among representative groups was similar, although linoleic acid was slightly reduced as a function of aging and the development of CHD. These results demonstrate that studies which measure only the absolute changes in FFA levels as a function of age or development of CHD tend to underestimate the magnitude of changes in FFA availability to tissues and for participation in biochemical reactions.

ESR studies of the interaction of copper(II)GHK, histidine, and Ehrlich cells.

The tridentate complex CuGHK does not form ESR detectable adducts upon addition to either glutathione or Ehrlich ascites cells under our conditions. The absence of adducts is consistent with the poor uptake of CuGHK by cells. ESR spectra are used to characterize adduct formation between CuGHK and histidine. The CuGHK-histidine adduct is not stable in the presence of Ehrlich ascites tumor cells. It is argued that a Cu(His)2 complex is formed as a consequence of the interaction of GHK with cells.

Free fatty acidemia as an inducer of systemic hyperfibrinogenemia and fibrinolytic inhibition.

Many major inflammatory stimuli induce secondary conditions of blood hyperfibrinogenemia and fibrinolytic inhibition, changes which may be mediated by alterations in free fatty acid (FFA) metabolism. The effect of a free fatty acidemia induced by the intravenous infusion of a triglyceride into rabbits on the fibrinogen/fibrinolytic system was determined. A 3-h infusion of synthetic fat emulsion induced a rapid rise in FFA (0.4-2.1 microEq/ml in 3 h) followed by a more gradual rise in fibrinogen (2.6-4.3 mg/ml at 24 h), alpha 1-antitrypsin (1.1-1.9 mg/ml at 48 h), and serum fibrinolysis inhibitory activity (increased 202% at 48 h). Increases in protein concentration were due to increased synthesis. It is proposed that the changes in the fibrinogen/fibrinolytic system which follow major inflammatory stimuli are induced by a mediating free fatty acidemia. Possible pharmacological procedures to block these changes are discussed.

Fatty acids, fibrinogen and blood flow: a general mechanism for hyperfibrinogenemia and its pathologic consequences.

Plasma fibrinogen is elevated in various stressful states and conditions in which active mobilization of free fatty acids (FFA) occurs. Reduction of plasma FFA by an assortment of hypolipidemic drugs is consistently followed by a decrease in the accompanying hyperfibrinogenemia. A direct link between FFA and fibrinogen has been demonstrated in animals, and in experiments employing incubated liver slices. Based on these clinical and experimental observations, we postulate that hepatic fibrinogen synthesis is stimulated by FFA. Since fibrinogen is a major determinant of whole blood viscosity, erythrocyte aggregation, and sludging of red cells in terminal and pre-terminal blood vessels, we propose that microcirculatory blood flow may be impaired in the presence of chronically elevated plasma FFA levls. Consequently, hypolipidemic drugs may be effective in prevention of circulatory complications associated with FFA-induced hyperfibrinogenemia.

Effects of glycyl-histidyl-lysyl chelated Cu(II) on ferritin dependent lipid peroxidation.

The copper binding tripeptide, glycyl-L-histidyl-L-lysine [GHK:Cu(II)] has a plethora of biological effects related to the wound healing process. The presence of iron complexes in damaged tissues is detrimental to wound healing, due to local inflammation, as well as microbial infection mediated by iron. To test if the wound healing properties of GHK:Cu(II) are due to an affect on iron metabolism, we examined the effects of GHK:Cu(II) on iron catalyzed lipid peroxidation. GHK:Cu(II) inhibited lipid peroxidation only if the iron source was ferritin. Whereas GHK:Cu(II) inhibited ferritin iron release it did not exhibit significant superoxide dismutase-like or ceruloplasmin-like activity. We propose that GHK:Cu(II) binds to the channels of ferritin involved in iron release and physically prevents the release of Fe(II). Thus, a biological effect of GHK:Cu(II), possibly related to wound healing, may be the inhibition of ferritin iron release in damaged tissues, preventing inflammation and microbial infections.

Suppression of acute-phase synthesis of fibrinogen by a hypolipidemic drug (clofibrate).

The effect of pretreatment of rats with a hypolipidemic agent, clofibrate ethyl alpha-(p-chlorophenoxy)isobutyrate on the acute-phase induction of fibrinogen synthesis by thrombin injections was evaluated. Rats were pretreated with graded dosages of clofibrate (10-300 mg/kg day), then injected intraperitoneally with 1,000 U topical thrombin at 0 hours and at 24 hours to induce an acute-phase induction of fibrinogen synthesis. In control rats, thrombin injections rapidly diminished plasma fibrinogen levels and induced a marked rise in serum free fatty acids. However, by 48 hours after the initial injection, fibrinogen biosynthesis increased 5.6 fold and plasma levels were elevated twofold above normal. In contrast, clofibrate pretreatment of rats markedly attenuated both the thrombin-induced free fatty acid mobilization and the subsequent stimulation of fibrinogen synthesis. The optimal dosage of clofibrate which blocked the effect of thrombin of fibrinogen synthesis was 100 mg/kg day. These results suggest that the injection of acute-phase enhancements of fibrinogen synthesis is mediated by prior alterations in free fatty acid metabolism and that hypolipidemic drugs which block FFA mobilization will also suppress acute-phase fibrinogen hyperproduction.

Evidence for a low-molecular-weight plasma peptide which stimulates chick chondrocyte metabolism.

Plasma contains a number of insulin-like activities (ILA) of molecular weights 7,000 to 90,000 (somatomedins and insulin-like proteins) which stimulate cellular metabolism and may function as growth factors. We have found evidence for the presence of an 800 Dalton peptide in human plasma which markedly stimulates the metabolism of chick chondrocytes. This peptide was extracted from human Cohn fraction IV-1 by procedures similar to those used for somatomedin isolations. At the Sephadex G-50 column separation step, the fraction with molecular weights of 300-1,000 was found to markedly stimulate chick chondrocyte metabolism. Rechromatography on Sephadex G-25 concentrated activity in peptides of molecular weight of about 800. An HPLC separation on a silica C-18 reverse phase column gave elution of the active peptide at 18% acetonitrile in water. This bioactivity appears to be a peptide which is free of lipids, carbohydrates, nucleic acids, metal ions, and immunoreactive insulin. This factor markedly increased the metabolism of cultured chick chondrocytes, but had only marginal activity on rat chondrocytes. When added at 1 microgram/ml to chick chondrocytes cultured in F-12 medium plus 1.5% fetal calf serum, the HPLC-purified activity increased DNA synthesis 7.3-fold, lipid synthesis 10.2-fold, and lactate production 2.9-fold after 48 h incubation. However, unlike somatomedins A and C, this factor did not displace insulin from placental membranes. These results suggest that low-molecular-weight peptides, which are smaller than the somatomedins, may contribute to the total ILA of human plasma.

The use of glycylhistidyllysine in culture systems.

Glycylhistidyllysine (GHL), a tripeptide isolated from plasma, has been shown to alter the growth rate of many cell types and organisms in culture systems. The tripeptide is optimally active at concentrations between 10 and 200 ng/ml. Some of the more interesting uses of GHL are highlighted in this paper. Present information suggests that GHL functions as a transporter of transition metals, in particular copper, to the cell surface for uptake into the cell.

Fat metabolism, the fibrinogen/fibrinolytic system and blood flow: new potentials for the pharmacological treatment of coronary heart disease.

Recent studies have emphasized the role of blood fibrinogen as a major determinant of blood and plasma viscosity in the microcirculation, of red cell and platelet aggregation, and in the growth of atheromatous lesions. Blood fibrinogen levels are increased and the endogenous fibrinolytic activity decreased by conditions or factors which raise plasma free fatty acid levels. Conversely, a wide variety of hypolipidemic agents lower fibrinogen levels and increase endogenous fibrinolytic activity apparently by reducing hepatic synthesis of fibrinogen and antifibrinolysins. These observations may open a new avenue for the pharmacological development of agents capable of improving the patterns of blood flow in patients with circulatory impairments.

Purification of growth-promoting peptides and proteins, and of histones, by high pressure silica gel chromatography.

A rapid method for the purification of histones and a variety of growth-promoting proteins and peptides by chromatography on silica gel has been developed. The isolation of the growth-promoting components of serum has been hampered by excessive losses associated with the use of water-based purification mens in acidic methanol-H2O solutions (eg. insulin, albumin, the somatomedins) provides a basis for purification on high-pressure silica gel columns, while peptides and histones can be purified in similar solvents. After column chromatography, the solvent is removed by flash-evaporation, or the protein may be precipitated directly from the solvent by neutralization of the pH and the addition of ethanol. The retention of biological activity (eg. somatomedin-C binding to insulin receptors and cell-growth stimulation) and recovery are excellent.

Growth-modulating tripeptide (glycylhistidyllysine): association with copper and iron in plasma, and stimulation of adhesiveness and growth of hepatoma cells in culture by tripeptide-metal ion complexes.

The tripeptide H-Gly-His-Lys-OH (GHL) is a human plasma constituent which has been previously shown to modulate the growth and viability of a variety of cell types and organisms. Experimental observations presented herein indicate that GHL is complexed with the transition metal ions Cu++ and Fe++ in vivo and may exert its biological effects as a peptide-metal chelate. At physiological pH in vitro, GHL associates with ionic copper, cobalt, iron, molybdenum, manganese, nickel, and zinc, but has no affinity for calcium, manganese, potassium, and sodium. GHL acts synergistically with copper, iron, cobalt, and zinc to alter patterns of cell growth in monolayer cultures of a tumorigenic hepatoma cell line (HTC4). These transition metals induce cellular flattening and adhesion to support surfaces, and inhibit DNA synthesis and lactic acid production when growth is limited by reduction of serum concentrations in medium. These inhibitory effects are neutralized, and intercellular adhesion and growth are stimulated by GHL in medium at nanomolar concentrations. Cu and Fe are the most active metals when combined with GHL. The results suggest that the inability of HTC4 cultures to replicate without adequate concentrations of serum in medium may reflect deficiency of GHL and transition metals, which appear to form complexes prior to interaction with cells. Chelation of transition metals with GHL and, potentially, with other growth-modulating peptide factors in plasma or medium, may provide a mechanism for expression and regulation of biological activities influenced by transition metals and polypeptide growth factors. The observed effects of GHL-metal complexes, including stimulation of cellular adhesiveness to substratum (flattening) and intercellular attachment (monolayer formation), appear to satisfy requirements for growth of hepatoma cells in monolayer culture.