Addition Of HDL Research Articles

Apolipoprotein A-I Stimulates Cell Proliferation in Bone Marrow Cell Culture.

Culturing of bone marrow cells in serum-free RPMI-1640 medium led to a decrease in the rate of DNA biosynthesis. Addition of HDL or their main protein component apolipoprotein A-I to the culture medium dose-dependently increased the rate of [3H]-thymidine incorporation into DNA. The maximum stimulation was achieved at HDL concentration of 80 μg/ml and apolipoprotein A-I concentration of 20 μg/ml. To identify the target-cells of apolipoprotein A-I, we used thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU) that incorporates into cell DNA at the stage of replicative DNA synthesis (S phase) and can be detected by fluorescence microscopy. In bone marrow cell culture, apolipoprotein A-I stimulates the proliferation of monocyte (monoblasts, promonocytes) and granulocyte (myeloblasts, promyelocytes) progenitor cells, as well as bone marrow stromal cells.

Abstract 180: Chronic Myelogenous Leukemia Cell Viability is Differentially Regulated by LXR Activation, HDL-associated Proteins, and LDL

LXR activation and HDL exert anti-cancer effects, albeit to varying degrees depending on cancer type and model. One of the mechanisms by which LXR and HDL impair cancer cell growth is through mediation of cellular cholesterol and phospholipid efflux, thereby limiting the availability of essential lipids for rapidly proliferating cells. Thus, the variability in LXR- and HDL-mediated anti-cancer effects may in part be due to differences in 1) HDL functionality and 2) the ability to induce expression of LXR target genes involved in lipid efflux and homeostasis due to adaptive shifts in cancer cell metabolism. Interestingly, serum amyloid A (SAA), a pro-inflammatory acute phase protein shown to be elevated in cancer, infection, and obesity, has recently been shown to differentially increase and decrease cholesterol efflux, despite its ability to displace apoA1 on HDL. Therefore, we investigated the effects of LXR activation, lipoproteins (HDL and LDL) and HDL-associated proteins (apoA1 and SAA) on the viability of chronic myelogenous leukemia cells - a cancer model previously unstudied in regards to cholesterol metabolism. Treatment of cells with the LXR agonist TO901317 (1μM, 5μM, and 10μM) significantly increased ABCA1 mRNA expression at 24hr and 72hr, in addition to dose-dependently decreasing HMG-CoA reductase and anti-apoptotic Bcl-xL mRNA expression. Changes in TO901317-induced gene expression corresponded to time- (24hr, 48hr, 72hr) and dose-dependent decreases in cell viability and average cell size. Interestingly, the addition of HDL to cell cultures with or without TO901317 treatment did not affect K562 cell viability, whereas LDL dose-dependently increased cell viability. Conversely, human-derived apoA1 (25 μg/mL) or HDL + apoA1 drastically reduced TO901317-treated K562 cell viability after 48hr and 72hr. K562 cell viability was modestly decreased by SAA (10 μg/mL) treatment, with greater decreases observed in cells treated with SAA + HDL. These findings suggest that LXR activation, HDL-associated proteins, and LDL differentially regulate chronic myelogenous leukemia cell viability.

High-density lipoprotein contribute to G0-G1/S transition in Swiss NIH/3T3 fibroblasts.

High density lipoproteins (HDLs) play a crucial role in removing excess cholesterol from peripheral tissues. Although their concentration is lower during conditions of high cell growth rate (cancer and infections), their involvement during cell proliferation is not known. To this aim, we investigated the replicative cycles in synchronised Swiss 3T3 fibroblasts in different experimental conditions: i) contact-inhibited fibroblasts re-entering cell cycle after dilution; ii) scratch-wound assay; iii) serum-deprived cells induced to re-enter G1 by FCS, HDL or PDGF. Analyses were performed during each cell cycle up to quiescence. Cholesterol synthesis increased remarkably during the replicative cycles, decreasing only after cells reached confluence. In contrast, cholesteryl ester (CE) synthesis and content were high at 24 h after dilution and then decreased steeply in the successive cycles. Flow cytometry analysis of DiO-HDL, as well as radiolabeled HDL pulse, demonstrated a significant uptake of CE-HDL in 24 h. DiI-HDL uptake, lipid droplets (LDs) and SR-BI immunostaining and expression followed the same trend. Addition of HDL or PDGF partially restore the proliferation rate and significantly increase SR-BI and pAKT expression in serum-deprived cells. In conclusion, cell transition from G0 to G1/S requires CE-HDL uptake, leading to CE-HDL/SR-BI pathway activation and CEs increase into LDs.

Abstract 520: High-density Lipoprotein Inhibits Human M1 Macrophage Polarisation through the Redistribution of Caveolin-1

Macrophages play a critical role in the development and progression of atherosclerosis. Depending on their surrounding milieu, macrophages can adopt a wide range of functional phenotypes; pro-inflammatory (M1) and pro-resolving (M2). HDL has many cardio-protective properties including potent anti-inflammatory effects, largely through the removal of cholesterol from cells. It is currently not known if this extends to influencing human macrophage phenotypes. Thus, we aimed to investigate the effect of HDL on human macrophage polarisation. Human blood monocyte-derived macrophages were induced to either an M1-phenotype by incubation with LPS and IFN-γ or to an M2-phenotype with IL-4. Macrophages were differentiated in the presence or absence of human HDL and their phenotypes were characterised using cell surface markers, reactive oxygen species (ROS) production by flow cytometry, and mRNA expression by real-time PCR. Downstream signalling pathways were also explored. We discovered that HDL inhibited the induction to M1 as evidenced by a decrease in cell surface marker expression; CD192 and CD64. This was accompanied by a decreased expression of M1-associated inflammatory genes TNF-α, IL-6 and MCP-1. However, HDL had no effect on induction to the M2 phenotype. Similarly, methyl-beta-cyclodextrin (MβCD), a non-specific cholesterol acceptor was also able to suppress M1 induction suggesting cholesterol efflux is important in this process. Further we found that HDL decreased membrane caveolin-1 in M1 macrophages and redistributed it intracellularly. The requirement of caveolin-1 was revealed as bone marrow-derived macrophages from Cav-1-/- mice continued to differentiate into M1 despite the addition of HDL. Moreover, we demonstrated a decrease in STAT3 and ERK1/2 phosphorylation in M1 macrophages treated with HDL, suggesting cholesterol efflux inhibits the STAT3s and MAPKs during induction to the M1 phenotype. Finally, we found that HDL also inhibited M1 function; with reduced reactive oxygen species (ROS) production. We provide evidence that HDL reduces macrophage induction to the inflammatory M1 phenotype, but not M2, via cellular redistribution of caveolin-1 and inactivation of STAT3 and ERK1/2 signalling pathway.

HDL protects against ischemia reperfusion injury by preserving mitochondrial integrity

ObjectiveHigh density lipoproteins (HDL) protect against ischemia reperfusion injury (IRI). However the precise mechanisms are not clearly understood. The novel intrinsic prosurvival signaling pathway named survivor activating factor enhancement (SAFE) path involves the activation of tumor necrosis factor (TNF) alpha and signal transducer and activator of transcription 3 (STAT3). SAFE plays a crucial role in cardioprotection against IRI. We propose that HDL protect against IRI via activation of the SAFE pathway and modulation of the mitochondrial permeability transition pore (mPTP) opening. Methods and resultsIsolated mouse hearts were subjected to global ischemia (35 min) followed by reperfusion (45 min). HDL were given during the first 7 min of reperfusion. In control hearts, the post-reperfusion infarct size was 41.3 ± 2.3%. Addition of HDL during reperfusion reduced the infarct size in a dose-dependent manner (HDL 200 μg protein/ml: 25.5 ± 1.6%, p < 0.001 vs. control). This protective effect was absent in TNF deficient mice (TNF-KO) or cardiomyocyte-STAT3 deficient mice (STAT3-KO). Similarly, HDL, given as a preconditioning stimulus, improved cell survival and inhibited mPTP opening in isolated cardiomyocytes subjected to simulated ischemia. These protective responses were inhibited in cardiomyocytes from TNF-KO and STAT3-KO mice. ConclusionOur data demonstrate that HDL protect against IRI by inhibition of mPTP opening, an effect mediated via activation of the SAFE pathway.

Advanced Glycation in macrophages induces intracellular accumulation of 7-ketocholesterol and total sterols by decreasing the expression of ABCA-1 and ABCG-1

BackgroundAdvanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7alpha- hydroxycholesterol and 7beta- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1.MethodsTotal cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot.ResultsIn control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages.ConclusionIn macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus.

Exogenous l-Arginine and HDL Can Alter LDL and ox-LDL-Mediated Platelet Activation

The aim of this study is to investigate the effects of exogenous L-arginine and HDL on LDL and oxidized LDL (ox-LDL)-mediated platelet activation. Adenosine diphosphate (ADP)-activated platelets have been incubated with lipoproteins with or without L-arginine. P-selectin receptor numbers per platelet have been measured by flow cytometry. After incubation with only L-arginine (without lipoproteins), platelet nitric oxide (NO) levels and P-selectin receptor numbers significantly increased compared to the controls (P < .05). After incubation with LDL or ox-LDL, receptor numbers of P-selectin significantly increased (P < .001). However, P-selectin receptor numbers in platelets treated with L-arginine + LDL or L-arginine + ox-LDL decreased compared to the levels in platelets treated with only LDL or ox-LDL (P < .01, P < .001, respectively). Addition of HDL to L-arginine + ox-LDL caused significant reduction in P-selectin receptor numbers as in the control values (P < .001).We have concluded that L-arginine causes enhanced platelet NO levels and blocks the effects of LDL or ox-LDL on platelet P-selectin receptor numbers and HDL also strengthens this effect of L-arginine.

HDL antielastase activity prevents smooth muscle cell anoikis, a potential new antiatherogenic property

Various studies using proteomic approaches have shown that HDL can carry many proteins other than its constitutive apolipoprotein A-I (apoA-I). Using mass spectrometry and Western blotting, we showed the presence of alpha(1)-antitrypsin (AAT) (SERPINA1, serpin peptidase inhibitor, clade A, an elastase inhibitor) in HDL, isolated either by ultracentrifugation or by selected-affinity immunosorption using an anti-apoA-I column. Furthermore, we report that HDL possesses potent antielastase activity. We further showed that only HDL but not LDL is able to bind AAT. HDL-associated AAT was able to inhibit extracellular matrix degradation, cell detachment, and apoptosis induced by elastase in human vascular smooth muscle cells (VSMCs) and in mammary artery cultured ex vivo. Degradation of fibronectin by elastase used as a marker of pericellular proteolysis was prevented by addition of HDL. Elastase present in aortic abdominal aneurysm (AAA) thrombus samples was also able to induce apoptosis of VSMCs in culture. This phenomenon was prevented by addition of HDL but not of LDL. Finally, we report that the proportion of AAT in HDL isolated from patients with an AAA is decreased relative to that from matched control subjects, suggesting a reduced capacity of HDL to inhibit elastase in these patients. In conclusion, our data provide evidence of a new potential antiatherogenic property of HDL attributable to AAT and its antielastase activity.

Rat Leydig cells use apolipoprotein E depleted high density lipoprotein to regulate testosterone production.

Rat HDL are known to increase testosterone production by cultured Leydig cells either following gonadotropin stimulation or cholesteryl ester depletion. However, rat HDL contain apolipoprotein E and have a high affinity for the members of the low density receptor family such as LDL receptor, LDL receptor related protein and VLDL receptor. In contrast with the adrenal cells, the contribution of apo A-I and apo E pathways in HDL cholesterol uptake has not been yet evidenced in rat Leydig cells. Recent data provided evidence that hCG stimulates scavenger receptor BI expression in testes. In order to investigate if testosterone production can be stimulated by apo E depleted HDL, we compared the level of testosterone stimulation by HDL with or without apo E first, in presence of saturating dose of hCG (1 IU/ml) and second, after depletion of cholesterol synthesis by pravastatin, an inhibitor of HMG-CoA reductase. In presence of hCG, HDL with or without apo E increased testosterone production respectively by 37 and 25%. Pravastatin at 100 microg/ml inhibited the cholesterol synthesis and the testosterone production by 25% and decreased the cholesteryl content by 25%. The addition of HDL with or without apo E (50 microg protein HDL/ ml) completely overcame the depletion of cellular cholesteryl esters and the inhibition of testosterone production induced by pravastatin. In the presence of heparin, apo E depleted HDL overcame the testosterone production induced by pravastatin, indicating that uptake of HDL without apo E via a secretion of apo E by the cells themselves was not involved. Therefore, in absence of apo E, it is suggested that rat Leydig cells used HDL to regulate steroidogenesis via an apolipoprotein A-I pathway.

Strong synergistic anti-peroxidative effects of HDL 3 and ascorbic acid against copper-catalyzed LDL peroxidation

The aim of this study was to investigate the effects of high density lipoprotein 3 (HDL 3) and ascorbic acid (AsA) in combination on copper-catalyzed low density lipoprotein (LDL) peroxidation. LDL and HDL 3 were isolated from sera of healthy volunteers. LDL protein, 200 μg/ml, was incubated in phosphate-buffered saline (PBS) containing 2.5 μM CuSO 4 in the absence or presence of AsA, with HDL 3 protein alone, or with coincubation of HDL 3, 200 μg/ml, and AsA, 20 μg/ml, at 37°C for up to 24 h. As a control, the same amount of control LDL protein was added to PBS. The protective effects of the HDL 3 and AsA were examined by both electrophoresis and determination of the lipid hydroperoxide (LPO) level in each sample. The concentration of AsA was also measured in samples containing AsA. The coincubation of HDL 3 and AsA exerts more powerful anti-peroxidative effects against copper-catalyzed LDL peroxidation, than either of these agents alone. In addition, AsA was retained in the media by the addition of HDL 3. The findings suggest that there are strong synergistic anti-peroxidative effects of HDL 3 and AsA and these two may act in concert in vivo to inhibit LDL peroxidation and thus exert an anti-atherosclerotic effect.

Association of estrogens with human plasma lipoproteins: Studies using estradiol-17β and its hydrophobic derivative

Estrogen replacement therapy is widely used to combat symptoms of menopause, but factors influencing the transport of estrogen in plasma and its cellular uptake have been explored only to a limited extent. In this study, labeled estradiol-17β (E 2) was compared with its hydrophobic derivative (estradiol-3,17-diacetate, E 2AA) in terms of (1) distribution within various lipoproteins and lipoprotein-free fractions of human plasma by ultracentrifugation and (2) uptake by human endothelial cells. Although added E 2 was predominantly bound to HDL and lipoprotein-free fractions, E 2AA was associated to some degree with VLDL and LDL but was still present in significant amounts in HDL and lipoprotein-free fractions. Significant associations of E 2 with lipoproteins were also confirmed by polyacrylamide gel electrophoretic separation of E 2-labeled plasma. In normal plasma, however, < 10% of E 2 was associated with lipoproteins when measured by radioimmunoassay. Incubation of E 2AA and E 2 with human dermal capillary endothelial cells showed similar uptake by 24 hours. A significant portion of E 2AA was hydrolyzed to estradiol-17-onoacetate both in plasma and in cells. The addition of HDL and LDL to medium containing lipoprotein-deficient serum significantly reduced labeled E 2 and E 2AA uptake, respectively, by endothelial cells. These studies suggest that (1) although the association of E 2 with HDL under normal conditions may be small, it increases significantly with higher estrogen concentrations of E 2; (2) the hydrophobic ester of E 2 shows increased binding to VLDL and LDL; and (3) this approach may be useful in manipulating the delivery of selected E 2 derivatives to cells.

HDL 3 Activates Phospholipase D in Normal but Not in Glycoprotein IIb/IIIa-Deficient Platelets

Glycoprotein IIb/IIIa has been proposed as the platelet receptor for high density lipoproteins (HDL 3). We characterized the HDL 3-induced second messenger response in normal and glycoprotein IIb/IIIa-deficient platelets. In normal platelets physiological concentrations of HDL 3 induced the time-dependent generation of phosphatidic acid in the absence of phosphoinositide turnover. The rise in phosphatidic acid preceded that of diacyglycerol which was inconsistent with phospholipase C/diacylglycerol kinase pathway being the source of phosphatidic acid and suggested the involvement of phospholipase D. In the presence of butanol, HDL 3 stimulated the accumulation of phosphatidylbutanol, an unequivocal indicator of phospholipase D activity. No increase in phosphatidic acid, diacylglycerol, and phosphatidylbutanol was observed upon addition of HDL 3 to glycoprotein IIb/IIIa-deficient platelets. We conclude that phosphatidic acid is generated in HDL 3-stimulated platelets by phospholipase D and that glycoprotein IIb/IIIa is the receptor involved in this process.

Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein.

Incubation of cocultures of human aortic endothelial (HAEC) and smooth muscle cells (HASMC) with LDL in the presence of 5-10% human serum resulted in a 7.2-fold induction of mRNA for monocyte chemotactic protein 1 (MCP-1), a 2.5-fold increase in the levels of MCP-1 protein in the coculture supernatants, and a 7.1-fold increase in the transmigration of monocytes into the subendothelial space of the cocultures. Monocyte migration was inhibited by 91% by antibody to MCP-1. Media collected from the cocultures that had been incubated with LDL induced target endothelial cells (EC) to bind monocyte but not neutrophil-like cells. Media collected from cocultures that had been incubated with LDL-induced monocyte migration into the subendothelial space of other cocultures that had not been exposed to LDL. In contrast, media from separate cultures of EC or smooth muscle cells (SMC) containing equal number of EC or SMC compared to coculture and incubated with the same LDL did not induce monocyte migration when incubated with the target cocultures. High density lipoprotein HDL, when presented to cocultures together with LDL, reduced the increased monocyte transmigration by 91%. Virtually all of the HDL-mediated inhibition was accounted for by the HDL2 subfraction. HDL3 was essentially without effect. Apolipoprotein AI was also ineffective in preventing monocyte transmigration while phosphatidylcholine liposomes were as effective as HDL2 suggesting that lipid components of HDL2 may have been responsible for its action. Preincubating LDL with beta-carotene or with alpha-tocopherol did not reduce monocyte migration. However, pretreatment of LDL with probucol or pretreatment of the cocultures with probucol, beta-carotene, or alpha-tocopherol before the addition of LDL prevented the LDL-induced monocyte transmigration. Addition of HDL or probucol to LDL after the exposure to cocultures did not prevent the modified LDL from inducing monocyte transmigration in fresh cocultures. We conclude that cocultures of human aortic cells can modify LDL even in the presence of serum, resulting in the induction of MCP-1, and that HDL and antioxidants prevent the LDL induced monocyte transmigration.

Behaviour of phospholipase modified-HDL towards cultured hepatocytes. II. Increased cell cholesterol storage and bile acid synthesis

Human total HDL (hydrated density 1.070–1.210), HDL 2 (1.070–1.125), HDL 3 (1.125–1.210) or HDL separated by heparin affinity chromatography were treated with or without purified phospholipase A 2 from Crotalus adamanteus. Control and treated HDL were reisolated and were then incubated with cultured hepatocytes. 1. Mass measurements evidenced a time-dependent cholesterol enrichment in hepatocytes cultured in the absence of lipoproteins. Addition of HDL 2 still enhanced by 25% the cell cholesterol content and down-regulated endogenous sterol synthesis in similar proportions. Conversely, HDL 3 slightly decreased the amount of free cholesterol in hepatocytes (−12%). 2. Incubations with phospholipase A 2-treated HDL resulted in a 35%–50% increase of both the cellular cholesterol esterification and the cholesterylester accumulation, when compared to cells cultured in the presence of control-HDL. This effect was observed with HDL 2, HDL 3 and combining the data with all subfractions. 3. Cultured hepatocytes secreted cholic and β-muricholic acids as major bile acids and HDL 2 showed a tendency to stimulate their secretion. Phospholipase treatment of HDL again induced an increased production by hepatocytes of those two bile acids. Thus, whereas HDL 2 and HDL 3 display different behaviours with respect to cell cholesterol content, neosynthesis and bile acid secretion, their modifications by phospholipases always orientate the cell sterol metabolism in the same direction: increased cholesterylester accumulation and bile acid production.

The role of lipoproteins in EBV early antigen induction in Raji cells

Synthesis of the Epstein-Barr virus (EBV) associated early antigen (EA) can be induced by a variety of agents in Raji cells, a latently EBV-infected Burkitt lymphoma line. We investigated the role of lipoproteins in this EA induction system. Cell growth was not affected by lipoprotein-deficiency, but EA induction by most combinations of the inducers TPA (tetradecanoyl-phorbol-acetate), IdU (iododeoxyuridine), n-BA (n-butyric acid), anti-IgM and EA inducing factor (EIF), was greatly reduced. Only the inducer combination TPA/n-BA was completely independent of the presence of lipoproteins, indicating a different induction pathway. Removing the lipid moieties of the culturing serum did not result in reduced EA induction. Thus, the lowered EA inducibility in lipoprotein-deficiency is due to the absence of the protein moiety (apolipoprotein). Addition of HDL or VLDL partially reconstituted the original EA inducibility, whereas LDL had no effect. Lipoproteins were particularly important during the first 4 hours of induction, the phase where inducers may act on cell membrane structures (e.g., receptors). But lipoproteins were also required throughout the incubation period, even in a late and inducer independent phase.

Activation of fibrinolysis by apolipoproteins of high density lipoproteins in man

The effects on fibrinolysis of purified normal human plasma lipoproteins and their apolipoproteins (apo) were assessed in an in vitro system containing urokinase, plasminogen, and fibrin. High and very low density lipoproteins (HDL and VLDL, respectively) but not low density lipoproteins (LDL) significantly increased lysis of radiolabeled fibrin or lysis area of plated fibrin compared to controls. Apo HDL, apo HDL 2, apo HDL 3, apo VLDL (buffer soluble apo), apo AI and AII activated fibrinolysis by 1.3 – 1.4 fold of control values, whereas apo B (as a narrow density cut of LDL) and non-lipoprotein control proteins (albumin and myoglobin) did not. Addition of HDL in the absence of urokinase failed to activate fibrin lysis. These data demonstrate that the major proteins of HDL — apo AI and apo AII — and possibly certain minor constituent(s) common to HDL and VLDL participate in the fibrinolytic process.

Dependence of human plasma lecithin: cholesterol acyltransferase activity upon high-density lipoproteins 2

The conclusion that HDL 2 suppress lecithin: cholesterol acyltransf erase activity has been re-examined using three assay methods. Removal by ultracentrifugation of lipoproteins of d 1.125 g/ml reduced activity to 53% ( n = 15) of control plasma. Replacement of VLDL and LDL was without effect, but addition of HDL 2 restored activity. Addition of HDL 2 to d 1.125 g/ml infranate in quantities greater than had been present in original plasma had no further effect on activity, and addition of HDL 2 to plasma had no effect on activity. It is concluded that HDL 2 are required for full expression of plasma lecithin: cholesterol acyltransferase activity.

Serum lipoproteins increase testosterone production in hCG-desensitized Leydig cells.

The effects of high density and low density lipoproteins (HDL and LDL) on testosterone synthesis by Leydig cells from rats treated three days earlier with 50 IU human chorionic gonadotropin (hCG) or from saline-injected control rats were investigated. HDL caused a significant, dose-dependent increase in both basal and hCG-stimulated testosterone production by Leydig cells from hCG-treated rats, but had no marked effect on Leydig cells from control rats. The serum testosterone concentration of hCG-treated rats was three times higher than in controls. Basal testosterone production by Leydig cells from hCG-treated rats was elevated two-fold in comparison to controls, while hCG-stimulated testosterone production was significantly lower than in controls. Both basal and hCG-stimulated testosterone production by Leydig cells from hCG-treated rats were significantly increased by the addition of HDL or LDL. Furthermore, HDL restored the hCG-stimulated testosterone production to the maximal amount produced by hCG-stimulated Leydig cells from control animals. When equivalent amounts of HDL or LDL cholesterol (140 microgram/ml) were added to the incubation, HDL caused a greater increase in testosterone production than did LDL. These results suggest that hCG-treatment, in vivo, caused a prolonged stimulation of the Leydig cells which resulted in depletion of the cholesterol available for steroidogenesis, since the addition of cholesterol in the form of HDL or LDL caused an increase in the testosterone production of these Leydig cells.

Effects of human plasma lipoproteins on platelet aggregation and platelet factor 3 activity in vitro

The effects of plasma lipoproteins on platelet function have been examined in vitro using platelet-rich plasma (PRP) and lipoproteins prepared from healthy normolipidaemic subjects. No spontaneous aggregation of platelets was induced by the addition of very low density (VLDL), low density (LDL) or high density (HDL) lipoprotein to PRP in amounts sufficient to raise the concentration by 2- to 7-fold. Incubation of PRP for 10–30 min at 37°C following equivalent increases in VLDL, LDL or HDL concentration had no effect on the subsequent aggregation of platelets by adenosine diphosphate (ADP), 1-epinephrine or collagen. Addition of HDL, but not of VLDL or LDL, to PRP or platelet-poor plasma was associated with a rise in platelet factor 3 (PF3) activity, as indicated by a shortened Stypven time. No lipoprotein altered the availability of PF3 in PRP, as measured by the reduction in Stypven time induced by kaolin or by freezing and thawing. These results indicate that the epidemiological relationships that exist between plasma lipoprotein concentrations and coronary heart disease, and the increases in platelet sensitivity to aggregating agents and in PF3 activity which have been observed in hyperlipidaemic subjects, are unlikely to be related to acute effects of lipoproteins on platelet function.