An improved method for detection of low density lipoprotein receptor defects in human T lymphocytes.

Familial hypercholesterolemia (FH) results from an inherited functional defect of the low density lipoprotein (LDL) receptor and is complicated by premature atherosclerosis. FH diagnosis is obtained by sophisticated techniques or is suggested by clinical criteria. We have developed a technique based on flow cytometry for the measurement of DiI-labeled LDL uptake in human peripheral blood T lymphocytes left for 2 days in a lipoprotein-deficient culture medium. Flow cytometry allowed us to discriminate the uptake of DiI-LDL by T lymphocytes subpopulation from the uptake by the whole mononuclear population using a T cell-specific anti-CD3 antibody. The method appeared to be highly specific for the receptor-mediated pathway of LDL uptake as DiI-LDL uptake was inhibited in the presence of a 10-fold excess of unlabeled LDL and by EDTA. A good relationship was found between the uptake of DiI-LDL and 125I-labeled LDL degradation. The test was applied in three groups of patients: patients with normal cholesterol levels, patients with heterozygous FH, and patients with high cholesterol levels but without clinical criteria of FH. The mean fluorescence intensities were 23.1 +/- 8.9, 6.3 +/- 1.7, and 17.1 +/- 3.5 (mean +/- standard deviation), respectively. The ability to measure the fluorescence in T lymphocytes improved the discrimination between FH and non-FH subjects when compared with values obtained from the whole mononuclear cell population. These results suggest that our method could be useful for LDL receptor defects screening.

Ciliation of bovine aortic endothelial cells in culture.

Solitary cilia were observed by electron microscopy in senescent bovine aortic endothelial cells in culture. Such single cilia have previously been seen in several tissues of various species, but as far as we know they have not been identified in cultured endothelial cells. The analysis of ultrathin sections enabled us to show that the cilia originated in one of the two centrioles. Vacuolar structures located at one end of the centrioles were also observed and might occur during the lengthening of the cilium. Moreover, the surface replication technique allowed us to show that the cilia could extend out of the cell. As senescent endothelial cells enter a quiescent stage, they could build up such a cilium as was observed for some strains of cultured fibroblasts.

Influence of a short oxidative stress on the LDL endocytosis by human endothelial cells: an ultrastructural study.

Following injury induced by oxidant stress (exposure to xanthine-xanthine oxidase during 120 min), cultured human umbilical vein endothelial cells were severely modified. These lesions were evaluated by electron microscopy and the types of injury were shown to be progressive cell blebbing as well as altered mitochondrial features. Enlargement of the endoplasmic reticulum and swelling of the Golgi apparatus were also observed. With the intention of learning more about the role of oxygen-derivative-induced alterations of the endocytotic apparatus (plasma membrane, endosomes, lysosomes), the receptor-mediated endocytosis of colloidal gold-conjugated LDL was evaluated. It was demonstrated that this endocytosis is drastically decreased following this type of injury, corroborating our previous report of important reduction in 125I-LDL endocytosis under the same conditions. Tubular electron-lucent structures, often observed in the vicinity of blebs, could possibly be involved in the impairment of LDL receptor accessibility.

Cytosolic free Ca2+ and proteolysis in lethal oxidative injury in endothelial cells.

Oxygen free radicals (OFR) are thought to mediate ischemia-reperfusion injury to endothelium of heart, lung, brain, liver, and kidney and contribute to development of atherosclerosis, pulmonary O2 toxicity, and adult respiratory distress syndrome. Increased cytosolic free Ca2+ (Cai2+) has been proposed as a mechanism of injury from oxidative stress, yet the pathways by which an increase in Cai2+ may cause OFR-mediated endothelial cell injury remain unknown. Using multiparameter digitized video microscopy and the fluorescent probes, fura-2 acetoxymethyl ester and propidium iodide, we measured Cai2+ and cell viability in human umbilical endothelial cells during oxidative stress with xanthine (50 microM) plus xanthine oxidase (40 mU/ml). Oxidative stress caused a sustained increase in Cai2+ from a resting level of 90-100 nM to near 500 nM, which was preceded by formation of plasma membrane blebs. The increase in Cai2+ was prevented by removal of extracellular Ca2+ (Cao2+). Prevention of the increase in Cai2+ was associated with prolonged cell viability. Readdition of Cao2+ resulted in an immediate large increase in Cai2+ and rapid onset of cell death. The protease inhibitors, leupeptin and pepstatin, delayed the increase in Cai2+ and prolonged cell viability. The results are consistent with the hypothesis that endothelial cell injury due to oxidative stress may be the result of Cai2+ influx and resultant activation of Ca(2+)-dependent proteases.

Endocytosis of low density lipoproteins in human endothelial cells: typical morphological aspects of the high affinity receptor-mediated pathway as revealed by serial sections and acid phosphatase cytochemistry.

We used electron microscopy serial sections and acid phosphatase cytochemistry to follow the endocytosis of low density lipoproteins (LDL) by cultured human umbilical vein endothelial cells. The surface LDL-receptors were labeled with LDL-colloidal gold conjugates at 4 degrees C and then, cells were incubated for 0, 5, 10, 20, 30 and 60 min at 37 degrees C before fixation. The organelles identified in this way and studied by serial sections corresponded to those described in endothelial cells and in other cell types, with a peripheral endosomal compartment and a juxtanuclear endosomal compartment, located in the vicinity of the Golgi apparatus and mainly composed of round vesicle-containing structures called multivesicular bodies. By acid phosphatase cytochemistry, the limits between the endosomal juxtanuclear compartment and the lysosomal compartment were also studied and it was shown that multivesicular bodies are probably the first site for the acquisition of acid hydrolases. Lastly, the observation of highly gold-labeled endothelial cells when cells were incubated for 90 min at 37 degrees C with the LDL-gold conjugate, suggested that LDL was degraded in the lysosomal compartment.

Ultrastructural, physico-chemical and conformational study of the interactions of gentamicin and bis(beta-diethylaminoethylether) hexestrol with negatively-charged phospholipid layers.

Aminoglycoside antibiotics such as gentamicin, which are fully hydrophilic, and cationic amphiphilic drugs such as bis(beta-diethylaminoethylether)hexestrol (DEH), are both known to inhibit lysosomal phospholipases and induce phospholipidosis. This enzymatic inhibition is probably related to the neutralization of the surface negative charges on which the lysosomal phospholipases A1 and A2 are dependent to express fully their activities (Mingeot-Leclerq et al., Biochem Pharmacol 37: 591-599, 1988). Using negatively charged liposomes, we show by 31P NMR spectroscopy that both gentamicin and DEH cause a significant restriction in the phosphate head mobility and, in sonicated vesicles, the appearance of larger bilayer structures. Both DEH and gentamicin increased the apparent size of sonicated negatively charged liposomes (but not of neutral liposomes) as measured by quasi-elastic light scattering spectroscopy. Examination of replicas from freeze-etched samples, however, revealed that gentamicin caused aggregation of liposomes, whereas DEH induced their fusion and the formation of intramembranous roundly shaped structures. Only DEH caused a significant decrease of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, a fluorescent lipid-soluble probe. In addition, DEH, but not gentamicin, interfered with the bilayer to hexagonal phase transition occurring in dioleoyl- and dielaidoylphosphatidylethanolamine liposomes upon warming, and caused the appearance of an isotropic signal suggestive of the formation of inverted micelles. In computer-aided conformational analysis of the molecules at a simulated air-water interface, gentamicin was shown to display a largely-open crescent shape. When surrounded by phosphatidylinositol molecules, it remained as such at the interface which it locally mis-shaped, establishing close contact with the negatively charged phospho groups. In contrast, DEH could be oriented perpendicularly to the interface, with its two cationic groups associated with the phospho groups, and its phenyl- and diethylethandiyl moieties deeply inserted between and interacting with the aliphatic chains. Thus, although both agents cause lysosomal phospholipases inhibition, the differences in their interactions with negatively-charged bilayers is likely to result in a different organization of the phospholipids accumulated in vivo, which could lead to different toxicities.

Incubation of endothelial cells in a superoxide-generating system: impaired low-density lipoprotein receptor-mediated endocytosis.

Endothelial cells (EC) of blood vessels are submitted to oxidative stress under various circumstances. These conditions may modify EC functions; therefore, in the present work we have studied the receptor-mediated endocytosis of low-density lipoproteins (LDL) and malondialdehyde-modified LDL by the LDL receptor and the "scavenger" receptor, respectively, in cultured human umbilical vein EC after short (0-120 minutes) incubations in a superoxide anion (O2-) generating system. In both receptor-mediated processes, the oxidative stress produces a significant decrease at four different LDL concentrations (5-50 micrograms/ml) after 120 minutes of oxidation. On the other hand, the fluid-phase endocytosis of sucrose by EC seems to be stimulated by these conditions. Furthermore, incorporation of antioxidant enzymes in the O2- -producing system shows that H2O2 is an obligatory intermediate in order to produce the effect on the receptor-mediated processes. Hypotheses concerning the mechanisms involved in the modifications of endocytotic processes and their implications in vivo are discussed.

Rapid preparative isolation of concentrated low density lipoproteins and of lipoprotein-deficient serum using vertical rotor gradient ultracentrifugation.

In order to study cellular metabolism of low density lipoproteins (LDL), ultracentrifugal methods have been used to isolate the lipoproteins. The use of vertical rotor ultracentrifugation very quickly produces small quantities of diluted lipoproteins per gradient, as well as small volumes of lipoprotein-deficient serum. We present modifications to this method in order to prepare routinely more concentrated LDL and a sufficient volume of lipoprotein-deficient serum in a relatively short time with minimal cost and handling.

Freeze-fracture study of isolated lysosomes of rat liver.

Freeze-fracture electron microscopy was utilized to pursue the morphological characterization of isolated lysosomes. Combining biochemical data of purity and morphological criteria of identification, it was possible to describe the convex and concave fracture faces of lysosomes. We have compared these with the corresponding faces of tritosomes i.e. lysosomes isolated after injection of Triton WR-1339 into the animal.

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient.

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.