Preclinical investigations of a medium-chain triglyceride:fish oil emulsion. I. Effects of bovine milk lipoprotein lipase on lipid composition

The bolus intravenous injection of a novel medium-chain triglyceride:fish oil emulsion (MCT:FO, 8:2, w:w) was recently found to increase within 60 min the leucocyte and platelet phospholipid content of long-chain polyunsaturated omega3 fatty acids. The present report deals with the effects of bovine milk lipoprotein lipase on the lipid composition of this emulsion. The results are compared to those obtained with either a pure fish oil emulsion or a medium-chain triglyceride: long-chain triglyceride:fish oil emulsion (MLF, 5:4:1). Emphasis is placed on i) differences in the fate of distinct fatty acids initially present in the triglycerides, di glycerides and phospholipids, ii) the generation of unesterified fatty acids relative to their initial content in each emulsion, and iii) the time course for these various events. The comparison between the three emulsions under consideration also provides information relevant to their respective sensitivity to lipoprotein lipase and suitability in terms of the generation of distinct unesterified fatty acids, including long-chain polyunsaturated omega3 fatty acids. Furthermore, attention is drawn to the greater efficiency for the hydrolysis of fatty acids from diglycerides as compared to triglycerides and a transient increase in the paired C8:0/C10:0 ratio in the diglycerides generated from the MCT:FO or MLF emulsion. The present study thus affords novel information relevant to the possible use of the MCT:FO emulsion in human subjects.     

Hemostatic safety of the bolus intravenous injection of a novel medium-chain triglyceride:fish oil emulsion

The bolus intravenous injection of a novel 8:2 medium-chain triglyceride:fish oil emulsion was recently found to increase within 60 min and for the subsequent 24-48 h the long-chain polyunsaturated omega3 fatty acid content of both leucocyte and platelet phospholipids in 12 normal subjects. The aim of the present report is to document the hemostatic safety of such a procedure in the same 12 subjects. No adverse effect was found when comparing the results obtained after administration of either the fish oil-containing emulsion or a control 5:5 medium-chain triglyceride:soybean triglyceride emulsion, whether in terms of the occlusion time in either an ADP or epinephrine test or in terms of the [CD]42b, [CD]62p, fibrinogen and PAC-1 response to ADP, collagen or thrombin receptor analog peptide 6 in platelets examined by fluorescence activated cell sorting. In conclusion, this novel procedure for the rapid enrichment of cell phospholipid in long-chain polyunsaturated omega3 fatty acids presents the required safety in a hemostatic perspective.     

Effects of a medium-chain triglyceride:fish oil emulsion administered intravenously to omega3 fatty acid-depleted rats on cationic fluxes in aortic rings

The handling of 45Ca and 86Rb by aortic rings obtained from rats depleted in long-chain polyunsaturated omega3 fatty acids (second generation) was examined in vitro over 10 to 60 min incubation at either increasing concentrations of extracellular K+ (5, 3 and 60 mM) in the case of 45Ca net uptake or in the absence and presence of ouabain (50 microM) in the case of 86Rb uptake. The omega3-depleted rats were injected intravenously 120 min before sacrifice with 1.0 ml of either an omega3 fatty acid-rich medium-chain triglyceride:fish oil emulsion (MCT:FO) or a control medium-chain triglyceride:olive oil emulsion (MCT:OO). In the MCT:OO-injected rats, the rise in extracellular K+ concentration failed to stimulate 45Ca net uptake, whilst the prior injection of the MCT:FO emulsion restored the expected increase in 45Ca net uptake by aortic rings exposed to 60 mM K+. The absolute value for 86Rb net uptake after 10 or 60 min incubation and whether in the absence or presence of ouabain, which significantly decreased the uptake of 86Rb+ after 60 min incubation, only represented in the MCT:FO-injected rats 63.1+/-3.8% of the mean corresponding values found in MCT:OO-injected animals. These findings are consistent with the view that activity of cationic channels, such as the voltage-sensitive Ca2+ channel, the outflow of Ca2+ as mediated by either Na+-Ca2+ countertransport or a Ca2+-ATPase, the activity of Na+,K+-ATPase and the modality of K+ inflow by an oubain-resistant modality are all affected in aortic cells by the content of long-chain polyunsaturated omega3 fatty acids in membrane phospholipids.     

Rapid changes in liver lipid composition and pancreatic islet K+ handling and secretory behaviour provoked by the intravenous administration of a medium-chain triglyceride: fish oil emulsion to long-chain polyunsaturated omega3 fatty acid-depleted rats

Second generation rats depleted in long-chain polyunsaturated omega3 fatty acids are currently used as an animal model for the insufficient dietary supply of such fatty acids often prevailing in Western populations. The present study deals mainly with the effects of a novel medium-chain triglyceride: fish oil emulsion (MCT:FO), as compared to a control medium-chain triglyceride:olive oil emulsion (MCT: OO), administered as an intravenous bolus to the omega3-depleted rats 60-120 min before sacrifice upon selected biochemical and biophysical variables. The major findings consisted of a severe decrease of the omega3 fatty acid content of liver lipids in non-injected omega3-depleted rats and its partial correction after injection of the MCT:FO emulsion. The omega3-depleted rats also displayed liver steatosis, increased incorporation of long-chain polyunsaturated omega6 fatty acids in liver phospholipids and increased activity of liver Delta9-desaturase. As judged from the effects of ouabain upon 86Rb net uptake by isolated pancreatic islets, the activity of Na+,K+-ATPase was virtually abolished in the omega3-depleted rats. The latter defect was corrected by prior intravenous injection of the MCT:FO emulsion, this coinciding with suppression of the excessive secretory response to a number of insulin secretagogues otherwise observed in the islets of omega3-depleted rats injected or not with the MCT:OO emulsion.     

Electron microscopy of endothelial cells in culture: III. Freeze-fracturing

Summary Endothelial cells can be examined by freeze-fracturing and replication techniques performed on pellets of endothelial cells scraped off their culture substratum or, alternatively, cells grown on pieces of cover glass can be fractured as monolayers. Deep etching can be performed on either preparation provided that the cryoprotectant glycerol is omitted and water, which sublimes away during the etching process, is substituted.The cleaning and handling of the replicas for subsequent viewing in the electron microscope must be performed with care and are described in detail.     

Regulation of hydrogen peroxide generation in cultured endothelial cells.

Endogenous hydrogen peroxide (H2O2) release from aortic endothelial cells was studied in the presence of antioxidant enzyme inhibitors, mitochondrial inhibitors, a microsomal cytochrome P-450 inhibitor, and after oxidative stress induced with H2O2 or menadione. Extracellular H2O2 generation was determined spectrofluorometrically using 3-methoxy-4-hydroxy phenylacetic acid, and intracellular H2O2 production (in or near peroxisomes) was measured indirectly using aminotriazole, which inactivates catalase in the presence of H2O2. Extracellular H2O2 release was 0.079 +/- 0.005 nmol/min/mg protein in Hanks' balanced salt solution, was constant during a 120-min incubation period, and was not affected by the cell passage number. The half-life for catalase inactivation with aminotriazole was 23 min. Inhibition of catalase, glutathione reductase, or gamma-glutamylcysteine synthetase did not change the rate of extracellular release of H2O2. Furthermore, inhibition of the mitochondrial respiratory chain (rotenone, antimycin A) or microsomal cytochrome P-450 (8-methoxypsoralen) did not change extracellular H2O2 release or intracellular H2O2 production (at peroxisomes) by endothelial cells or cells in which glutathione reductase was inactivated. When the cells were exposed to exogenous H2O2 (30 microM), extracellular H2O2 was scavenged primarily by the glutathione redox pathway. Exogenously added H2O2 (100 microM) changed intracellular H2O2 production (in or near peroxisomes) only when the glutathione redox cycle was inactivated. Menadione (20 microM), which undergoes intracellular redox cycling, increased extracellular H2O2 release almost 4-fold to 0.3 nmol/min/mg protein. Furthermore, menadione increased peroxisomal H2O2 levels and decreased the half-life for catalase inactivation in the presence of aminotriazole to 13 min. Catalase inhibition increased extracellular H2O2 release during menadione treatment, indicating that H2O2 can diffuse across the plasma membrane during oxidant stress.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanosine induces necrosis of cultured aortic endothelial cells.

We have observed that treatment of cultured bovine aortic endothelial (BAE) cells with guanosine can inhibit the proliferation and viability of the cells. The addition of 500 mumol/L guanosine to the medium resulted in approximately 90% inhibition of cell proliferation. It also changed the morphology of BAE cells from having a small cobblestone-like appearance to a giant pancake-like morphology. At a concentration range of 1 to 2.5 mmol/L, guanosine inhibited the viability of quiescent BAE cells. Incubation of the cells with 2 mmol/L guanosine for 24 hours maximally induced the loss of cell viability by approximately 80%. We also compared the effects of different nucleosides on the proliferation and viability of BAE cells and found that at appropriate concentration ranges, only guanosine was able to inhibit the proliferation and viability of the cells. To assess the mechanism that mediates the cytotoxicity of guanosine, we analyzed the degradation pattern of DNA in guanosine-treated cells and found that random DNA degradation occurred in the cells. Thus, we suggest that treatment of BAE cells with guanosine induced cell necrosis.     

Uptake of 125I-labelled C3a by cultured human endothelial cells.

The interactions of C3a anaphylatoxin with vascular endothelium were studied in vitro using human endothelial cells in culture and 125I-labelled human C3a. Cultured endothelial cells took up 125I-C3a in a time- and concentration-dependent manner and inactivated it. Uptake was not associated with binding to specific receptors since the amount of radioactivity accumulated by the cells was not influenced by treatment with excess unlabelled peptide, metabolic inhibitors or by low temperature. Further, we observed that uptake was not saturated during 90 min of incubation or within the concentration range of C3a tested (10(-9)--10(-6) M). C3a was taken up more rapidly than other labelled, less basic compounds, including Tyr5-bradykinin, lysozyme and albumin. Examination of the cells by autoradiographic electron microscopy revealed labelled material within the cell cysoplasm but not within specific intracellular structures, such as vesicles or vacuoles. C3a was partially inactivated after incubation with endothelial cells for 15 min, but some spasmogenic activity was retained even after 90 min incubation. Since the peptide is readily inactivated by the cells, the radioactivity in the cytoplasm may be inactive C3a and possibly C3a fragments. The combination of uptake and inactivation of C3a by endothelial cells may be an effective means of removing the peptide from circulation.     

Sustained enrichment of liver phospholipids and triglycerides in eicosapentaenoate after a bolus intravenous injection of a medium-chain triglycerides:fish oil emulsion to streptozotocin (Type 1) and Goto-Kakizaki (Type 2) diabetic rats

This study deals with the sustained enrichment of liver phospholipids and triglycerides in long-chain polyunsaturated omega3 fatty acids (omega3) found after the bolus intravenous injection of a novel medium-chain triglyceride:fish oil emulsion (MCT:FO) to streptozotocin (Type 1) and Goto-Kakizaki (Type 2) diabetic rats. Twenty hours after injection of the MCT:FO emulsion, the relative concentration of omega3 was indeed higher in liver phospholipids and triglycerides than that found in rats injected with either saline or a control medium-chain triglyceride:long-chain triglyceride emulsion. This coincided with a decrease in the ponderal percentage of C18:3omega3, C20:4omega6 and/or C22:4omega6 in liver triglycerides. The present study further documents differences between streptozotocin-induced and Goto-Kakizaki diabetic rats in terms of body weight, glycemia, liver triglyceride content and the fatty acid pattern of both liver phospholipids and triglycerides, as well as a close correlation in the latter animals between liver and plasma phospholipids or triglycerides as far as the ratio in the relative concentration of selected fatty acids representative of desaturase and elongase activities is concerned. In light of these and previous findings, it is proposed that the beneficial metabolic and functional events of the MCT:FO emulsion may display not solely a rapid but also sustained time course.     

Effect of hyperoxia on the cytoarchitecture of cultured endothelial cells.

When confluent pulmonary artery endothelial cells in culture were exposed to hyperoxia (95% O2 and 5% CO2), they became enlarged and mean corpuscular volume increased 30-35%. Rhodamine-phalloidin staining of actin filaments demonstrated that hyperoxia was associated with a progressive alteration in the actin distribution. Three days after oxygen exposure, the number and thickness of cytoplasmic stress fibers were increased, while the peripheral bands were disrupted or absent. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the amount of filamentous actin was increased in oxygen-exposed cells, while the total actin content remained unchanged, suggesting that oxygen exposure shifted the equilibrium from G actin to F actin.     

Cellular alterations in cultured endothelial cells exposed to therapeutic ultrasound irradiation.

Restoration of blood supply to tissue with impaired perfusion depends on spontaneous or mediated angiogenesis, which among other mechanisms includes stimulation, migration, and proliferation of endothelial cells (ECs). Therapeutic ultrasound (US) irradiation is known as an inducer of cellular modifications and is used to accelerate wound healing. An in vitro setup was developed in order to allow for a comprehensive investigation of cellular alterations induced in cultured ECs after exposure to different modes of therapeutic US irradiation. Viability assays revealed a higher rate of proliferation in the sonicated groups, although cell death was not observed. Visualization of actin stress fibers demonstrated partial disassembly of the fibers immediately after US sonication, with a maximum after about 2 h. However, 24 h following sonication the fibers regain normal appearance. A similar behavior was observed with the microtubules and focal adhesion complexes. Utilizing a wound healing assay revealed that migration rate of ECs is enhanced by US irradiation. These findings hint that therapeutic US sonication of ECs results in temporarily cellular alterations, which may induce tissue remodeling via stimulation of EC proliferation and migration.     

Binding of Escherichia coli S fimbriae to cultured human endothelial cells.

The binding of Escherichia coli adhesins to human umbilical vein endothelial cells was studied by a cell monolayer enzyme-linked immunosorbent assay. S fimbriae displayed a concentration-dependent and saturable binding to the endothelial cells which was mediated by their sialylgalactoside-specific lectin activity. P fimbriae exhibited only low binding, and type 1 fimbriae exhibited no binding to these cells.     

Reversible stimulation of lymphocyte motility by cultured high endothelial cells: mediation by L-selectin.

Lymphocyte emigration from blood into peripheral lymph nodes is mediated by specialized high endothelial cells (HEC) lining the postcapillary venules. A current model for this process postulates that it occurs in three steps: weak, selectin-mediated interactions tether lymphocytes to the blood vessel wall; the lymphocytes are activated to increase the affinity of integrin-dependent adhesion and enhance motility; and finally the lymphocytes migrate actively across the endothelial cell layer. Some features of this model are simulated in vitro by cultured HEC, which support the adhesion and transmigration of lymphocytes. In particular, cultured HEC stimulate lymphocytes to change shape from spherical to polar. This shape change provides a convenient assay of the motility activation of lymphocytes. In this paper it is shown that this occurs without the lymphocytes becoming tightly adherent, but depends on contact with the endothelial cell surface. The shape change is labile: non-adherent polar lymphocytes removed from HEC revert to round with a half-time of less than 8 min. Reagents which block the interaction of L-selectin with its ligands inhibit the HEC-induced shape change; these include mannose-6-phosphate, fucoidan, polyphosphomannan ester, treatment of HEC with sialidases and an anti-L-selectin monoclonal antibody known to block its lectin function. The change in shape is partially inhibited by antisera to the L-selectin ligand GlyCAM-1. Thus it is concluded that in this in vitro system, L-selectin-mediated binding of lymphocytes to HEC is essential for optimal induction of the shape change. Lymphocytes change shape in response to cultured HEC without loss of surface L-selectin, although activation stimuli are known to promote shedding of neutrophil L-selectin as well as motility and increased adhesiveness. However, the lymphocyte change in shape is a reversible process, and this may have implications for the nature and sequence of the signals transmitted from endothelium to lymphocytes during homing to peripheral lymph nodes.     

Group B streptococci invade endothelial cells: type III capsular polysaccharide attenuates invasion.

Group B streptococci (GBS) are the most common cause of neonatal sepsis and pneumonia. The pathogenesis of GBS disease is not completely defined. GBS-induced endothelial cell injury is suggested by histological findings at autopsy and in animal studies. We hypothesized that (i) type III GBS (COH-1) invade and injure human umbilical vein endothelial (HUVE) cells and (ii) isogenic mutations in GBS capsule synthesis would influence HUVE invasion. Confluent HUVE monolayers were infected for 0.5, 2, or 6 h. Media with penicillin plus gentamicin were added and incubated for 2 h to kill extracellular bacteria. Cells were washed and lysed, and the number of live intracellular bacteria was determined by plate counting. COH-1 invaded HUVE cells in a time-dependent manner at levels 1,000-fold higher than those of the noninvasive Escherichia coli strain but significantly lower than those of Staphylococcus aureus. There was no evidence for net intracellular replication of GBS within HUVE cells. COH-1 infection of HUVE cells caused the release of lactate dehydrogenase activity. GBS invasion was inhibited by cytochalasin D in a dose-dependent manner; GBS-induced lactate dehydrogenase release was attenuated by cytochalasin D. The isogenic strains COH 1-11, devoid of capsular sialic acid, and COH 1-13, devoid of all type III capsule, invaded HUVE cells three- to fivefold more than the parent COH-1 strain. The type III capsular polysaccharide and particularly the capsular sialic acid attenuate GBS invasion of HUVE cells. Electron micrographs of lung tissue from a GBS-infected newborn Macaca nemestrina also showed GBS within capillary endothelial cells. We conclude that endothelial cell invasion and injury are potential mechanisms in the pathogenesis of GBS disease.     

Replication of dengue and junin viruses in cultured rabbit and human endothelial cells.

The flavivirus dengue and the arenavirus Junin are both associated with a hemorrhagic shock syndrome in man. We have demonstrated the replication of these viruses in vitro in both rabbit and human endothelial cells by viral titers and immunofluorescent antibody studies. Rabbit endothelium established in continuous culture was derived from vena cava, while human cells in primary culture were derived from umbilical veins. In rabbit endothelium, dengue-2 virus passaged through monkey kidney monolayer cells (LLC-MK2) or human lymphoblastoid cells (raji) produced significantly more virus than the seed obtained from suckling mouse brain (MB). Inoculation of actively dividing, subconfluent human endothelial cells with the LLC-MK2 degue virus produced higher viral titers than inoculation of confluent cells. The appearance of Junin virus was delayed beyond that of dengue virus in rabbit endothelial cells although equivalent titers of virus were produced. In human cells, Junin virus was less productive than dengue virus and produced characteristic cycles of virus release. This is the first direct evidence for replication of human hemorrhagic fever viruses in endothelial cells.     

Atherogenic levels of low-density lipoprotein increase endocytotic activity in cultured human endothelial cells.

Cultured human umbilical vein endothelial cells (EC) exposed to atherogenic low-density lipoprotein (LDL) levels for protracted periods demonstrated heightened endocytosis. Confluent EC were incubated with LDL 90 to 240 mg/dl cholesterol for 1 to 4 days and endocytosis was measured by 14C-sucrose uptake. Control EC and cells incubated with 90 mg/dl LDL cholesterol showed similar uptakes of 14C-sucrose during all measured time periods. In contrast, EC exposed to 240 mg/dl LDL cholesterol showed an increase in endocytosis beginning at 2 days, whereas 160 mg/dl LDL cholesterol promoted increased uptake by 4 days. The endocytotic activity of LDL-perturbed EC is reduced to levels seen in control cells by cytochalasin B, an actin polymerization inhibitor. This finding suggests a modulatory role for the cytoskeleton in endocytosis changes. Examination of LDL-perturbed EC cytoskeleton reveals structural remodeling resulting in a marked increase in stress fibers. Cytochalasin B exposure causes a loss of stress fibers with the formation of globular filamental aggregates. Such LDL-induced cellular functional changes may contribute mechanistically to endothelial dysfunction, which is widely held to be a major contributing factor in the pathogenesis of atherosclerosis.     

Anti-CD14 antibodies reduce responses of cultured human endothelial cells to endotoxin.

Lipopolysaccharide (LPS) activates both myeloid and endothelial cells. Whereas CD14 has been shown to be involved in LPS recognition by myeloid cells, the mechanism responsible for the strong response of endothelial cells to LPS remains to be elucidated. The role of CD14 in this process was studied using CD14-specific antibodies (Ab). Anti-CD14 Ab inhibited LPS-induced interleukin-6 (IL-6) release and E-selectin expression by cultured human umbilical vein endothelial cells (HUVEC). Messenger RNA encoding IL-6 and E-selectin was reduced in parallel. The inhibitory effect of anti-CD14 Ab was epitope dependent, maximal at low LPS concentrations and dropping with increasing LPS doses. Anti-CD14 Ab did not affect endothelial cell activation induced by IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) and phorbol 12-myristate 13-acetate (PMA). IL-6 release and E-selectin expression of HUVEC were strongly reduced when LPS activation was performed in the absence of serum, indicating involvement of serum components in LPS activation of HUVEC. Nevertheless, anti-CD14 Ab also blocked LPS-induced HUVEC activation in the absence of serum. Although the role of serum components in LPS activation remains to be elucidated, CD14 seems to be a key mediator in LPS-induced activation of endothelial cells.     

Minimally oxidized low-density lipoprotein induces tissue factor expression in cultured human endothelial cells.

Oxidatively modified low-density lipoprotein is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis through its biologic effects on vascular cells. This study examined the effects of minimally oxidized preparations of LDL (MM-LDL) on tissue factor (TF) expression by cultured human endothelial cells. Low-density lipoprotein purified from normal donors was modified by exposure to iron or by prolonged storage, resulting in levels of thiobarbituric acid-reacting substances of approximately 2.5 to 4 nmoles/mg cholesterol. Preparations had less than 2.5 pg of endotoxin per microgram LDL and had no intrinsic procoagulant activity. This form of modified but not native LDL induced TF expression in endothelial cells in a time- and dose-dependent manner. Peak TF coagulant activity in cells exposed to 40 micrograms/ml MM-LDL were observed at 4 to 6 hours, and ranged from 50 to 500 pg/10(5) cells, compared with less than 10 pg/10(5) cells exposed to native LDL. Northern blot analysis showed TF mRNA levels to increase approximately 30-fold with exposure to MM-LDL for 2 hours. Induction of TF activity was dependent on the concentration of MM-LDL from 1 microgram/ml to 80 micrograms/ml, a range in which cell viability and morphology were unaffected. The findings suggest that minimally oxidized LDL may be a local mediator promoting thrombosis in atherosclerotic lesions.