Zbigniew Rymaszewski

Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or Cu+2 ions: Identification of major products and their effects

Oxidation of lipoproteins is believed to play a key role in atherogenesis. In this study, low density lipoproteins (LDL) was subjected to oxidation in the presence of either human umbilical vein endothelial cells or with Cu+2 ions and the major oxides formed were identified. While cholesterol-alpha-epoxide (C-alpha EP) was the major product of cholesterol peroxidation in the presence of endothelial cells, cholest-3,5-dien-7-one (CD) predominated in the presence of Cu+2 ion. Both steroids were identified by gas chromatography/mass spectrometry. HDL cholesterol was resistant to oxidation. When tested on human skin fibroblasts in culture C-alpha EP (10 micrograms/ml) caused marked stimulation of 14C-oleate incorporation into cholesterol esters, while CD stimulated cholesterol esterification only mildly. These studies show that a) C-alpha EP is the major peroxidation product of LDL cholesterol moiety in the presence of endothelial cells and b) it causes marked stimulation of cholesterol esterification in cells. C-alpha EP may play a key role in increasing cholesterol esterification noted in atherogenesis.

Estimation of cellular DNA content in cell lysates suitable for RNA isolation

Methods presently available for the isolation of RNA are incompatible with conditions necessary for the measurement of either DNA or cell number, resulting in infrequent quantitation of messenger RNA in relation to the quantity of cells studied. In the present studies, a microfluorometric method has been modified from previous techniques to permit the quantitation of DNA in cell lysates prepared using an acid guanidinium thiocyanate-phenol (AGTP) solution from which RNA can subsequently be isolated. The lysate is incubated in alkaline EDTA, then neutralized with KH2PO4, followed by the addition of the fluorochrome bisbenzimidazole (Hoechst 33258), and measurement of fluorescence. DNA content is comparable in measurements by the present technique and by the diphenylamine method on parallel samples. DNA content per cell for human cells measured with this technique is comparable to that previously reported using other methods. The use of AGTP solution results in stability of measurable DNA in cell lysates for periods of at least 10 weeks (permitting batching of samples and retrospective measurement) and stability of fluorescence for at least 20 h after the addition of bisbenzimidazole making the timing of fluorescence measurement less critical. The technique described should permit quantitation of messenger RNA in relation to DNA (and hence indirectly to cell number) on a routine basis.

Human retinal vascular cells differ from umbilical cells in synthetic functions and their response to glucose

Abstract Cell culture systems have commonly been used to study mechanisms implicated in the pathogenesis of diabetic retinopathy, but the great majority of cell preparations used have been either of nonhuman retinal origin or nonretinal human origin. Because of questions of species and organ specificity in the function of cells of vascular origin, in this study, cultured microvascular endothelial cells (HREC), pericytes (HRPC), and pigment epithelial cells from the postmortem human retina, and endothelial cells from human umbilical vein (HUVEC) were evaluated with respect to cell proliferation, and secretory products potentially important in diabetic retinopathy, i.e., prostaglandins (PG) and plasminogen activators (PA), normalized to DNA content/well, under both basal (5 mM) and high (25 mM) glucose conditions. Glucose (25 mM) reduced DNA content similarly in both types of endothelial cells, had a lesser effect on HRPC, and did not significantly alter the proliferation of pigment epithelial cells. Basal secretion of PGI2 (measured as 6-keto-PGF1α) was in the order HRPC ≫ HREC > HUVEC, whereas PGE2 secretion was in the order HREC ≫ HRPC > HUVEC. Glucose (25 mM) stimulated PGI2 secretion by HRPC, but not by either type of endothelial cell, and enhanced PGE2 secretion by HREC, but not by HUVEC or HRPC. Release of plasminogen activator activity differed between HUVEC and HREC under basal conditions and addition of 25 mM glucose stimulated release only from HREC. Glucose (25 mM) stimulated PA secretion by HREC, but not by HUVEC. These findings provide evidence that human retinal pericytes are an important source of prostacyclin, and that there are differences between HREC and HUVEC with respect to secretory functions and their modulation by glucose, indicating regional specificity of these functions, Extrapolation to human retinal vascular cells from experiments using cells from heterologous vascular beds to draw inferences about the pathophysiology of diabetic retinopathy are not valid for these cellular functions.

Menadione-Induced Oxidative Stress in Bovine Heart Microvascular Endothelial Cellsa

Objective: Oxidative stress from increased production of reactive oxygen species or decreased efficiency of inhibitory and scavenger systems may contribute to vascular injury. In this study, we developed an in vitro model of vascular injury by menadione‐induced oxidative stress in bovine heart microvascular endothelial cells.

Cholestyramine treatment in early life of low-density lipoprotein receptor deficient Watanabe rabbits: decreased aortic cholesteryl ester accumulation and atherosclerosis in adult life.

Effect of cholestyramine treatment in early life of Watanabe heritable hyperlipidemic rabbits (an animal model lacking low-density lipoprotein receptor activity) on subsequent (6 months recovery) occurrence of natural atherosclerotic lesion and arterial cholesterol metabolism was investigated. Initial cholestyramine treatment decreased both plasma total cholesterol and HDL-cholesterol levels which normalized within 4 weeks after treatment was discontinued. At 9 months of age (age of occurrence of spontaneous atherosclerotic lesions), the extent of aortic atherosclerosis in cholestyramine pre-treated animals was modestly lower (P less than 0.05), as compared to controls, with a significant (P less than 0.05) decrease in aortic cholesteryl ester content. Furthermore, at the end of the recovery period aortic activity of acyl-CoA: cholesterol acyltransferase and neutral cholesterol esterase activity was significantly (P less than 0.05) lower in cholestyramine-pretreated animals. These studies show that early cholestyramine pre-treatment in a low-density lipoprotein receptor-deficient animal model causes persistent changes which might influence cholesteryl ester accumulation and atherogenesis in adult life, even after cholestyramine treatment is discontinued.

High Acyl-CoA cholesterol acyl transferase activity in fetal rabbit aorta: Evidence for the presence of stimulating factor(s) in amniotic fluid

The activity of Acyl CoA-cholesterol acyl transferase was markedly high in fetal aortas when compared to maternal and adult male rabbits. This activity dropped by 50% at 1 week of age. This high activity in fetal aorta a) did not appear to be due to changes in plasma cholesterol levels or to the later development of endogenous inhibitor in the aorta, but rather b) due to stimulatory factor(s) present in amniotic fluid.

Studies on aorta during development. II. Differences in ontogeny of the key enzymes involved in cholesteryl ester synthesis and hydrolysis in rabbit aorta.

It is well known that cholesteryl ester accumulation is dramatically increased in the atherosclerotic artery. The enzymes acyl-CoA: cholesterol acyltransferase (ACAT), acid cholesteryl esterase (ACE) and neutral cholesteryl esterase (NCE) may play key roles in the accumulation of cholesteryl esters in the arterial wall. However, very little is known regarding the developmental pattern of the key enzymes involved in cholesteryl ester synthesis and hydrolysis. The total activities of ACAT, ACE and NCE were measured by radioassay using liposomal substrates in rabbit aortic homogenates. Our results indicate that ACAT activity decreases as a quadratic function with age (P less than 0.05). ACAT activity (pmol/100 mg protein/min) decreased from a high value in the fetus at term (63.3 +/- 7.4) to gradually lower values with increasing age. On the other hand, ACE activity (pmol/mg protein/min) was low in the fetus at term, and changed as a quadratic function with age (P less than 0.05) increasing gradually to higher activities with age up to a maximum at 12 weeks then decreased at 21 weeks. NCE activity (pmol/mg protein/min) increased dramatically from a low value in the fetus at term (3.34 +/- 0.48) to a maximum value at 1.5 weeks (14.65 +/- 2.73) then decreased as a linear function with increasing age up to 21 weeks (P less than 0.05). Plasma total cholesterol (mg/dl) also increased sharply from the fetal value at term of 98.5 +/- 5.2 to a maximum value at 1.5 weeks of 666.4 +/- 33.4, then decreased as a quadratic function with increasing age up to 21 weeks (40.8 +/- 6.7) (P less than 0.05). The free cholesterol content (microgram/mg protein) of the aortic tissue was initially high in the fetus (24.8 +/- 5.9) then increased with age. Examination of the ratio of synthesis to hydrolysis of cholesteryl esters as an index of enzyme activity units demonstrated a very high index in the fetus of 6.1 that rapidly decreased with increasing age in the young adult rabbit down to a value of 0.4 by 21 weeks of age. Correlation coefficients between enzyme activities, plasma cholesterol levels and aortic cholesterol levels indicated (a) a positive correlation of NCE activity with plasma cholesterol, (b) a negative correlation of NCE and ACE with aortic-cholesteryl ester content, and (c) no significant correlation of ACAT activity with either plasma cholesterol or aortic cholesterol content, indicating other factors are involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholestyramine treatment in early life: Immediate and delayed effects on arterial cholesteryl ester metabolizing enzymes in the rabbit☆

Feeding of cholestyramine-enriched diet to weaned normocholesterolemic rabbits resulted in: lowering of plasma cholesterol and distinctly decreased activity of aortic acyl-CoA cholesterol acyl transferase with no changes in aortic acid and neutral cholesteryl esterase activity. At 9 weeks after cessation of cholestyramine treatment enhanced activity of both aortic esterases were noted despite normalization of plasma cholesterol. No evidence for the presence of plasma factor influencing esterases activity was found in lipoprotein-free serum from cholestyramine-treated animals. These studies show that cholestyramine treatment in early life causes immediate and delayed changes in rabbit arterial cholesteryl ester metabolizing enzymes.

Inhibition of nucleoside transport by reactive oxygen species in bovine heart microvascular endothelial cells.

Cellular homeostasis depends on the structural and functional integrity of the membrane bilayer. Damage to the membrane can interfere with vital cellular processes, including signal transduction, molecular recognition, maintenance of the membrane potential, cellular metabolism and transport of molecules. Modification of the membrane bilayer by reactive oxidative species (ROS) is a major contributor to membrane damage and has been implicated in many pathological processes. In a number of diseases, injury to endothelial cells is mediated by oxidative species generated during ischemia/reperfusion or by activated neutrophils. During ischemia and oxidant injury, several metabolic events occur, including depletion of intracellular ATP and formation of a number of purine catabolites including inosine (Ino), hypoxanthine (Hyp) and adenosine (Ado) (Halliwell and Gutteridge, 1990). Accumulated Hyp can be oxidized by xanthine oxidase when the tissue is oxygenated, causing rapid generation of Superoxide and hydrogen peroxide. On the other hand, formation of Ado is especially important because it has vasodilatory and antiinflammatory activity. Alterations in nucleoside transport (NT) by ROS might have an effect on extracellular Ado concentration in vivo. Reduced Ado transport could elevate Ado concentrations, especially when Ado is formed extracellularly. Inhibition of NT could also diminish extracellular nucleoside uptake required for the reconstitution of intracellular nucleotide pools after cellular stress. Decreased nucleoside efflux might protect cells by allowing more efficient intracellular nucleoside salvage.

Human Breast Milk Stimulates Bile Acid Secretion by Fetal Rabbit Liver in Organ Culture

Abstract Fetal livers from rabbits at 30 days of gestation were grown in organ culture and the effect of human milk added to the culture medium on the ability of liver to excrete bile acids (cholylglycine) was examined. Human breast milk promoted a concentration related increase in cholylglycine accumulation in the medium. The factor(s) present in milk responsible for this effect appear to be non-protein in nature and is associated with the floating lipid fraction. Furthermore, milk enhances the integrity of liver explants, as established by light microscopy.