Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

The role of bovine lipoproteins in the regulation of steroidogenesis and HMG-CoA reductase in bovine adrenocortical cells.

The sources of cholesterol for steroid hormone production were examined using bovine adrenocortical (BAC) cells in primary culture. The experiments were designed to determine the effects of lipoproteins on cortisol production and the level of BAC cell 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Most studies on BAC cell lipoprotein requirements have been conducted using human low-density lipoprotein (hHDL); none have used the homologous bovine lipoproteins. BAC cells treated with corticotropin (ACTH) in a medium devoid of lipoproteins increased and maintained cortisol production 7- to 20-fold above basal levels. Under such conditions ACTH also increased the rate of HMG-CoA reductase activity. Inhibition of HMG-CoA reductase with mevinolin inhibited cortisol production by 85%, indicating that the cells were using cholesterol synthesized de novo for steroid production. Cortisol production was increased almost 40-fold above basal levels if hLDL (100 micrograms/ml) was included in the incubation medium. Human LDL also suppressed the levels of HMG-CoA reductase in a concentration-dependent fashion. Human HDL was without effect on either BAC cell steroidogenesis of HMG-CoA reductase. Addition of bovine LDL (bLDL) to the incubation medium also caused an increase in cortisol production and inhibited cholesterol synthesis. By contrast to hHDL, bHDL (100 micrograms/ml) increased the ability of BAC cells to produce cortisol production. Bovine HDL (bHDL) also was able to decrease HMG-CoA reductase, but not to the extent caused by hLDL or bLDL. These data demonstrate that bovine adrenal cells can use bHDL as a source of cholesterol for steroid hormone production. These findings may be of particular importance when one considers that in vivo, the bHDL content of bovine serum greatly surpasses the level of bLDL.     

Multiple effects of serum low-density lipoprotein on cultured human fibroblasts.

The effects of serum low-density lipoproteins (LDL) were studied in cultures of human skin fibroblasts grown in medium supplemented with human serum deficient in lipoproteins and in platelet factor. The LDL led to a temporary increase in the rate of cell replication, to increases in the cell content of protein and cholesterol, to an increase in average cell size, and to an increased secretion of glycosaminoglycans. The increases in cholesterol and protein were proportional to the increase in cell size, suggesting that the additional protein and cholesterol were of a structural, rather than a storage, nature. The increase in cell protein during the first few days of exposure to LDL was due to a decrease in the rate of protein degradation. Ultrafiltration of the serum to remove substances of molecular weight less than 30,000 did not reduce the basal rate of cell proliferation but did prevent the stimulation of proliferation by LDL; it did not alter the effect of LDL on cell protein and cholesterol, indicating that the latter responses are independent of the mitogenic action. The response of cells from diabetic donors did not differ from that of normal cells.     

Serum-induced leakage of negatively charged liposomes at nanomolar lipid concentrations

An enzyme inhibition assay was developed to determine methotrexate-gamma-aspartate leakage from liposomes at lipid concentrations as low as 43 nM phospholipid. When negatively charged liposomes prepared with phosphatidylglycerol/cholesterol 67:33 or phosphatidylinositol/cholesterol 67:33 were incubated in 10% (v/v) newborn calf serum, they leaked over 90% of their contents in 2 min. In contrast, liposomes prepared from phosphatidylcholine/cholesterol 67:33 leaked 18% of their contents under the same conditions. The amount of negative charge required to induce liposome leakage was determined by preparing liposomes with varying amounts of phosphatidylglycerol and phosphatidylcholine. Extensive leakage was observed only from liposomes prepared with greater than 50 mol of phosphatidylglycerol per 100 mol of phospholipid. The effect of the phase transition temperature on leakage of negatively charged liposomes in 10% (v/v) serum was investigated by using a series of phosphatidylglycerols with varying acyl chain lengths. Liposomes prepared from distearoylphosphatidylglycerol or dipalmitoylphosphatidylglycerol leaked less than 18% of their contents in 10% serum, whereas liposomes prepared with dilauroylphosphatidylglycerol or unsaturated lipids leaked more than 70% of their contents. Lipoprotein removal from serum followed by treatment with lipid to remove residual apoproteins reduced the leakage from phosphatidylglycerol liposomes in 10% serum. Phosphatidylglycerol liposomes leaked 73% in the presence of human low-density lipoproteins, but only 29% in the presence of bovine apolipoprotein A-I, and 25% in the presence of human high-density lipoproteins. Phosphatidylglycerol/cholesterol and phosphatidylserine/cholesterol liposomes leaked 67% in 4 mg/mL bovine serum albumin purified by cold ethanol extraction. The leakage of liposomes in albumin solutions could be substantially reduced by treating the albumin with lipid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular aging-dependent decrease in cholesterol in membrane microdomains of human diploid fibroblasts

Recently it has been shown that cholesterol plays indispensable roles in the function of cholesterol-rich microdomains (rafts), such as in ligand-mediated signal transduction. Using a perfringolysin O derivative (BCtheta) that binds selectively to cholesterol in rafts without causing membrane damage (Proc. Natl. Acad. Sci. USA 98 (2001) 4926), we have investigated the effect of in vitro replicative aging of human diploid fibroblasts, TIG-1, on the distribution of plasma membrane cholesterol. The amount of BCtheta-labeled membrane cholesterol decreased during replicative aging of TIG-1 cells, whereas total cholesterol increased somewhat. The relationship was confirmed by double staining with BCtheta and senescence-associated-beta-galactosidase, a biomarker of senescent cells. Cell fractionation experiments revealed decreases in both cholesterol in rafts and a raft marker, flotillin, during replicative aging. In addition, hydroxyurea-induced prematurely senescent cells also showed a lower level of BCtheta-labeled cholesterol than untreated cells, despite maintaining the total amount of cholesterol. When TIG-1 cells were cultured in cholesterol-deficient medium, BCtheta labeling was first diminished and then premature senescence was induced. Taken together with the reduced signaling capacity of aged cells, these results suggest that plasma membrane cholesterol in raft microdomains is more sensitive to senescence than total cholesterol and is a primary target in aging.     

Growth factor profile of irradiated human dermal fibroblasts using a serum-free method

Radiation therapy for cancer permanently damages tissue in the line of treatment. This study sought to establish a serum-free protocol to evaluate the growth of irradiated fibroblasts and to analyze the levels of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) compared with normal fibroblasts. One irradiated cell line of human dermal fibroblasts was established from an intraoperative specimen obtained from a patient who had undergone radiation therapy for head and neck cancer. Irradiated and normal fibroblasts were then plated in UltraCULTURE (serum and growth factor free), modified Webber's medium (bFGF 50 ng/ml, insulin-like growth factor 100 ng/ml), and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (serum with undefined basal growth factors). Irradiated cells were also seeded in UltraCULTURE with 50 and 100 ng/ml of bFGF. Cell counts were performed at 0, 1, 3, 5, and 7 days, and cell supernatants were assayed for bFGF and TGF-beta. Irradiated and normal fibroblasts exhibited stronger growth in modified Webber's medium than in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum. Growth of irradiated fibroblasts under bFGF modulation was similar to their growth in Webber's medium. Furthermore, irradiated fibroblasts remained viable in a serum-free and growth factor-free environment for at least 7 days; however, their growth and autocrine growth factor production was less than that of normal cells. This confirms the results of previous studies suggesting that cells from irradiated tissue undergo cellular changes. This study provides an effective model for the first-line evaluation of agents to improve wound healing, and it helps to establish standard levels of bFGF and TGF-beta production for irradiated fibroblasts.     

Lipoprotein-mediated efflux of radiolabeled cholesterol from cells does not indicate net removal of cellular cholesterol mass

The efflux of cholesterol from human skin fibroblasts was determined using radioisotope techniques and mass measurements. When the cells were labeled with [14C]- or [3H]-cholesterol and then incubated with very low density, low density, or high density lipoproteins or with serum, 20 to 30% of the label was released into the medium in 20 h. However, when the cellular cholesterol content was determined after incubation with various lipoproteins under identical conditions, only the heavier subfraction of high density lipoproteins (HDL3) caused a significant decrease in cellular cholesterol. This net removal of cholesterol can be observed in the cells without overloading them with cholesterol, by incubation with low density lipoproteins. Time studies indicated that at least 24 h of incubation is required to detect significant removal of cellular cholesterol. These experiments show that methods using the release of labeled cholesterol from cultured cells to determine net cholesterol removal mediated by high density lipoprotein, although currently used by many investigators, can lead to erroneous conclusions when employed without the measurement of cholesterol mass.     

Lipoproteins increase growth of mitogen-stimulated arterial smooth muscle cells

The relationship between lipoproteins and growth of aortic smooth muscle cells has been a matter of controversy. We therefore reexamined this issue using serum-free defined media methodology. By themselves, LDL or HDL (50-500 micrograms/ml) from normolipemic human or bovine plasma produced little or no growth of homologous aortic smooth muscle cells incubated in serum-free medium that was supplemented with insulin and transferrin to maintain cell viability. In fact, LDL prepared in the absence of an antioxidant (BHT) was toxic to these cells. However, in the presence of maximally effective concentrations of platelet-derived growth factor (PDGF), LDL or HDL consistently increased the growth of homologous smooth muscle cells (up to twofold increased in DNA accumulation in 48 hr). Lipoproteins also augmented the growth response of arterial smooth muscle cells to fibroblast growth factor or epidermal growth factor. The mechanism of this effect was investigated further with HDL, because, in contrast to LDL, HDL apoproteins are water-soluble. Neither HDL delipidated by solvent extraction (apoHDL), purified bovine apoA-I, nor cholesterol added in the form of phospholipid vesicles appreciably increased PDGF-induced growth of bovine smooth muscle cells. However, HDL-like particles reconstituted by sonication of apoHDL with cholesterol and phospholipids did increase the growth of cultures of bovine smooth muscle cells treated with PDGF. Uptake of tritiated thymidine by cultures incubated with partially purified PDGF alone (10 micrograms/ml) was 5,693 +/- 235 dpm/24 hr compared to 10,381 +/- 645 dpm/24 hr (p less than 0.01) in the presence of both PDGF and reconstituted HDL-like particles (250 micrograms protein/ml). Thus both the lipid and protein components of HDL may be necessary for optimal potentiation of growth of mitogen-stimulated cells. These results indicate that lipoproteins from normolipemic sera are not bona fide growth factors but can potentiate the growth of mitogen-stimulated cells, perhaps by supplying exogenous cholesterol required for membrane biogenesis. This finding might be important in arterial injury when the release of PDGF and exposure to plasma lipoproteins could act in concert to stimulate the proliferation of smooth muscle cells.     

Promotion of human T lymphocyte activation and proliferation by fatty acids in low density and high density lipoproteins

Mitogen-induced lymphocyte DNA synthesis measured by [3H]thymidine incorporation and lymphocyte proliferation assessed by counting the number of cells were reduced by greater than 95% when cells were cultured at low density in the absence of serum. Supplementation with either transferrin or lipoprotein alone only partially restored lymphocyte responses. Addition of both transferrin and lipoproteins of each major subclass permitted mitogen-induced lymphocyte DNA synthesis and proliferation equal to that observed in serum-containing medium. The degree of enhancement was dependent on the concentration of the lipoprotein added and could not be explained by the nonspecific addition of protein to the defined medium. The mechanisms of growth promotion by various lipoprotein fractions did not appear to be explained by provision of cholesterol to the cells. Neither cholesterol nor cholesteryl ester from endogenous sources or supplied exogenously was able to enhance mitogen-induced lymphocyte responses. In contrast, fatty acids, phospholipid, and triglyceride alone supported lymphocyte responses. Furthermore, lipoproteins retained the capacity to enhance lymphocyte responses following extraction of neutral lipid. Both low density lipoprotein and high density lipoprotein, subclass 3, increased the number of cells initially activated by mitogenic stimulation and supported the subsequent continued growth of the activated cells. Low density lipoprotein was more efficient than high density lipoprotein, subclass 3, in this latter regard. These results indicate that lipoproteins can promote maximal growth of mitogen-activated lymphocytes in transferrin-containing medium by providing growth factors other than cholesterol necessary for initial activation and required for continued lymphocyte proliferation.     

Seminal plasma choline phospholipid-binding proteins stimulate cellular cholesterol and phospholipid efflux

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa, collectively called BSP proteins) that potentiate sperm capacitation induced by high-density lipoproteins. We showed recently that BSP proteins stimulate cholesterol efflux from epididymal spermatozoa and play a role in capacitation. Here, we investigated whether or not BSP proteins could stimulate cholesterol and phospholipid efflux from fibroblasts. Cells were radiolabeled ([3H]cholesterol or [3H]choline) and the appearance of radioactivity in the medium was determined in the presence of BSP proteins. Alcohol precipitates of bovine seminal plasma (designated crude BSP, cBSP), purified BSP-A1/-A2, BSP-A3 and BSP-30-kDa proteins stimulated cellular cholesterol and choline phospholipid efflux from fibroblasts. Efflux mechanistic differences were observed between BSP proteins and other cholesterol acceptors. Preincubation of BSP-A1/-A2 proteins with choline prevented cholesterol efflux, an effect not observed with apolipoprotein A-I. Also, the rate of BSP-induced efflux was rapid during the first 20 min, but leveled off thereafter in contrast to a relatively slow, but constant, rate of cholesterol efflux mediated by apolipoprotein A-I, apolipoprotein A-I-containing reconstituted lipoproteins (LpA-I) and high-density lipoproteins. These results indicate that fibroblasts are a good cell model to study the mechanism of lipid efflux mediated by BSP proteins.     

Cytostatic effect of antiestrogens in lymphoid cells: relationship to high affinity antiestrogen-binding sites and cholesterol

The antiproliferative effect of 10(-6) M antiestrogens in an estrogen receptor-negative lymphoid cell line (K36) was enhanced in lipoprotein-poor growth medium. The enhancement was not due to increased bioavailability because cellular uptake of [3H]tamoxifen was not increased and the lipoprotein fraction of serum had negligible [3H]tamoxifen-binding capacity. Cholesterol and lipoproteins, but not mevalonate, reversed the cytostatic effect of antiestrogens. Reversal by cholesterol was dose-related (10(-7) M to 10(-5) M), while that by lipoproteins could also be demonstrated in medium undepleted of lipoproteins. The cytostatic efficacy of a series of ten compounds correlated well with their relative binding affinities for solubilized antiestrogen-binding sites from K36 cells when log IC50 values (concentration required to reduce [3H]thymidine incorporation by 50%) were plotted against log RBA50 values (concentration required to reduce [3H]tamoxifen binding by 50%) (correlation coefficient 0.94). Transmission electron microscopy of antiestrogen-treated cells showed evidence of disordered cytokinesis which was partially reversed by cholesterol. These observations implicate the antiestrogen-binding protein in the antiproliferative effect of antiestrogens in nonestrogen target cells.     

Lipid and protein contributions to the membrane surface potential of vesicular stomatitis virus probed by a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin

The surface potential of membranes of vesicular stomatitis virus and liposomes was determined by shift of ionization over a wide pH range of the membrane-inserted fluorophore, 4-heptadecyl-7-hydroxycoumarin. Incorporation into sonicated vesicles of negatively charged phosphatidylserine markedly increased the surface potential of uncharged phosphatidylcholine, but no significant effect on surface potential was produced by polar but uncharged glucocerebroside incorporated in phosphatidylcholine vesicles. The membrane of vesicular stomatitis virus was found to have a moderately high surface potential. Contributing to this viral membrane surface potential were glycoprotein spikes and phospholipid headgroups as determined by lowered charge after treatment of intact virions with thermolysin to remove glycoprotein or phospholipase C to remove phospholipid headgroups. The role of viral glycoprotein was confirmed by demonstrating increased surface charge of vesicles reconstituted with both viral glycoprotein and lipids compared with vesicles reconstituted with viral lipids alone. An unexpected finding was the large contribution to surface potential of cholesterol present in viral membrane. Increasing cholesterol concentration in virions by interaction with cholesterol-complexed serum lipoproteins resulted in a marked decrease in surface potential, whereas 75% depletion of virion cholesterol by interaction with sphingomyelin-complexed serum lipoproteins resulted in a significant increase in virion membrane surface potential. Although removal of glycoprotein spikes or depletion of cholesterol causes reduction in infectivity of vesicular stomatitis virus, no direct correlation could be found between alteration in surface charge and infectivity.     

Protein metabolism in the tumour-bearing mouse. Rates of protein synthesis in host tissues and in an Ehrlich ascites tumour at different stages in tumour growth.

The activity of acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) was measured in fibroblast homogenates from Niemann-Pick Type C (NPC) and Type D (NPD) patients to determine whether these cells exhibit similar defects in the regulation of cholesterol esterification. ACAT activity in normal cells cultured in the absence of serum lipoproteins responded rapidly (within 6 h) to the addition of serum and reached peak levels at 12-24 h, whereas little stimulation of activity in NPC cells was observed. In contrast, ACAT activity in NPD fibroblasts (cell lines from four different patients) began to increase between 6 and 12 h after serum addition, reaching levels up to 50% of normal values at 24 h. ACAT activity in NPC and NPD cell extracts could not be stimulated by preincubation with normal cell homogenates, nor was complementation between NPC and NPD homogenates observed. Addition of 25-hydroxycholesterol to fibroblasts cultured in delipidated serum increased ACAT activity for all three cell types, although stimulation in NPD cells was less than that observed in NPC cells. ACAT activity of deoxycholate-solubilized homogenates reconstituted into phosphatidylcholine vesicles was independent of the presence of serum lipoproteins during culture and dependent on cholesterol present in the vesicles for all cell types. However, ACAT activities of mutant fibroblasts in vesicles plus cholesterol were significantly (about 40%) lower than control levels. These results suggest that the metabolic lesions in NPC and NPD cells are biochemically distinct and that both may involve factors in addition to the availability of cholesterol substrate for the ACAT enzyme.     

The effects of phorbol ester on anabolism of lipids of cultured human skin fibroblasts

We have investigated the independent effects of phorbol ester (phorbol 12-myristate 13-acetate) on anabolism of the major lipid components in cultured diploid human skin fibroblasts. When we incubated these cells with [3H]acetate in serum-free medium for 18 h in the presence of 16 nM phorbol ester, [3H]acetate incorporation and the cellular content of cholesterol ester increased, and free cholesterol decreased. Enhancement of [3H]acetate incorporation into cholesterol ester was also observed when the cells were incubated with phorbol ester for 5 h in medium containing lipoprotein-deficient serum. Incorporation of [3H]galactose into glycosphingolipids increased many fold upon exposure of the cells either to fetal calf serum or separately to phorbol ester. Therefore, phorbol ester independently affects cholesterol and glycosphingolipid metabolism in a way that may be similar to that reported for serum low-density lipoproteins and unknown other factors in fetal calf serum. We have observed these effects of phorbol ester in the intact living cell. These findings should provide useful means for the study of metabolism of the plasma membrane lipid components in fibroblasts.     

Cholesterol as a limiting factor in the growth of Mycoplasma pneumoniae.

Ultracentrifugation was used to separate three commercial lots of bovine serum fraction (BSF) into components designed to contain lipoproteins. Each BSF lot and component was tested for ability to support the growth of tree strains of Mycoplasma pneumoniae. In general, the level of growth-promoting activity corresponded to the amount of cholesterol present in the BSF or BSF components rather than to the amount or type of lipoprotein Cholesterol was the limiting nutritional factor of BSF with low growth-promoting activity. The addition of cholesterol and bovine serum albumin to BSF with low activity resulted in growth equal to or greater than that observed for BSF with high growth-promoting activity. When cholesterol was added to agar medium containing BSF of low activity, mycoplasma colonies were greater in number, possessed larger mean diameters, and had centers that were more distinct than those observed when this BSF was used alone. Variability in growth-promoting actions of commercial lots of BSF was eliminated by increasing their cholesterol content to an optimum level. An adjustment of the cholesterol and albumin levels of any serum product used in culture media may provide a simple convenient method to improve growth and isolation of mycoplasmas.     

A comparative study of surface binding of human low density and high density lipoproteins to human fibroblasts: regulation by sterols and susceptibility to proteolytic digestion.

Binding of 125I-low density lipoprotein (LDL) and 125I-high density lipoprotein (HDL) was determined in cultured human fibroblasts from a normal subject and two subjects with homozygous familial hypercholesterolemia (HFH). Binding was assayed at 0 degree C to minimize the internalization of labeled lipoproteins. The binding of LDL and of HDL were compared following interventions reported to affect LDL binding in normal fibroblast. LDL binding to normal cells increased two to three fold 24 hours after transfer from medium containing whole fetal calf serum to medium containing lipoprotein-deficient fetal calf serum. This increase was completely blocked in the presence of cycloheximide (200 microgram/ml) or 7-ketocholesterol (2.5 microgram/ml). This increased capacity of normal fibroblasts to bind LDL could be reduced 70-80% by a subsequent 18-hour incubation with cholesterol (50 microgram/ml) or 7-ketocholesterol (2.5 microgram/ml). In contrast, no significant change in HDL binding to normal fibroblasts was observed after any of these interventions. HFH cells to show any significant change in either LDL binding or HDL binding following these interventions. These results suggest that HDL binding sites on normal fibroblasts are for the most part distinct from LDL binding sites. They also support the conclusion that LDL binding sites on HFH cells are for the most part qualitatively different from those on normal cells.     

Plasmodium falciparum: one-step growth in a semi-defined medium and the stimulatory effect of human seric lipoproteins and liposomes

The ring stages of Plasmodium falciparum within red blood cells cultured with complete medium stop growing when transferred to a basic medium containing RPMI plus fatty acid-free bovine serum albumin and dialyzable factors from human serum. Growth and multiplication can be partially restored by the addition of lipoprotein fractions prepared from human serum. No specificity was observed with subclasses of lipoproteins. Synthetic liposomes containing lecithin, oleic acid, and cholesterol mimic the effect of lipoproteins.     

Comparative studies on human skin fibroblasts: Life span and lipid metabolism in medium containing fetal bovine or human serum

Summary Three strains of human skin fibroblasts were cultivated in nutrient medium supplemented either with human serum or fetal bovine serum, and growth and lipid synthesis were compared. Rates of cellular growth were similar in both kinds of medium, but the replicative life spans of all three strains were curtailed significantly in human-serum medium. Incorporation of label into the major classes of neutral lipids from [¹⁴C]acetate and³H₂O was increased also in human-serum medium. Since human serum contained higher concentrations of cholesterol known to reduce endogenous cholesterol synthesis, these results were unexpected. Nonlipid factors in human serum may account for the shortened cellular life spans and increased lipogenesis and perhaps for the potential to develop atherosclerosis. Supported by grants from the Ontario Heart Foundation and Medical Research Council of Canada during the tenure of a Senior Research Fellowship from the Ontario Heart Foundation (J.T.C.) and a Scholarship from the Medical Research Council of Canada (S.G.).     

[14C]Acetate incorporation by cultured normal,familial hypercholesterolemia and Down's syndrome fibroblasts

Fibroblasts from patients with homozygous familial hypercholesterolemia (FH), a disease characterized by accelerated atherogenesis, are known to lack functional low-density-lipoprotein receptors, which ultimately results in increased cholesterol biosynthesis in the cultured cells. [¹⁴C]Acetate incorporation in these cells was compared to that of normal fibroblasts and to fibroblasts from patients with Down's syndrome, a disease in which atherosclerosis is rare. Total [¹⁴C]acetate incorporation did not differ significantly between normal and Down's fibroblasts, nor did its partitioning into the hexane-extractable and aqueous fractions of the cell hydrolysates. [¹⁴C]Acetate incorporation was much greater in FH cells in both the aqueous and hexane-extractable fractions. Preincubation in fetal bovine serum increased acetate incorporation only by FH cells, while 50 μg low-density lipoprotein/ml medium depressed acetate incorporation in all three groups. A C₂₇ sterol, identified by gas chromatography-mass spectrometry as a probable isomer of cholesterol, was present in small amounts in FH fibroblasts, but was not detectable in the normal or Down's cells. The absolute amounts of [¹⁴C]acetate incorporated into the non-sterol lipids were greater in the FH fibroblasts, indicating that these cells may have to synthesize, in addition to cholesterol, other required cellular lipids which are delivered to the normal cells by low-density lipoproteins.     

Characteristics of the cholesterol efflux induced by novel seminal phospholipid-binding proteins

Our recent results indicated that the major proteins of bovine seminal plasma (collectively called BSP proteins) stimulate cholesterol efflux from fibroblasts and that this process shows many differences compared to the efflux induced by apolipoprotein A-I (apoA-I)-containing lipoproteins. The present study was undertaken to investigate the BSP-mediated efflux mechanism. Compared to the slow and constant rate of cholesterol efflux induced by apoA-I-containing lipoproteins, the BSP proteins stimulated a rapid efflux that gradually reached a plateau. The addition of purified BSP proteins after the establishment of the plateau resulted in a further cholesterol efflux indicating that cellular cholesterol was still available for efflux. Incubation of unlabeled fibroblast culture with the spent medium containing BSP-generated lipid ([(3)H]cholesterol) particles obtained after the establishment of the plateau did not result in any cholesterol influx. Therefore, the plateau did not correspond to an equilibrium of the radiolabel between the medium and the cells but rather to a saturation of the efflux particles with cholesterol. Numerous studies have indicated that the cholesterol efflux induced by apoA-I-containing lipoproteins involves cell-surface receptor, caveolae and intracellular cholesterol mobilization. Therefore, we investigated these characteristics for the BSP-mediated cholesterol efflux. Binding of BSP proteins to cells (evaluated by immunoblotting) reached saturation rapidly and remained constant thereafter. However, after several washings the cell-bound BSP proteins were unable to promote significant cholesterol efflux. Both results indicate no correlation of cholesterol efflux with cell binding. Moreover, in comparison to apoA-I-mediated cholesterol efflux, BSP-mediated efflux was not abolished at temperatures below 22 degrees C indicating that the BSP-induced cholesterol efflux does not involve intracellular cholesterol mobilization. High-density lipoprotein- and apoA-I-mediated cholesterol efflux was inhibited by preincubating fibroblasts with progesterone, whereas the cholesterol efflux by BSP proteins was not, indicating that cell-surface caveolae do not participate in BSP-mediated cholesterol efflux. Our results indicate that the mechanism of cholesterol efflux by BSP proteins is unidirectional and is strikingly different from that mediated by apoA-I-containing lipoproteins.