Decrease in high density lipoprotein binding sites is associated with decrease in intracellular cholesterol efflux in dedifferentiated aortic smooth muscle cells.

One of the key features of atherosclerosis formation and progression is 'dedifferentiation' of contractile arterial smooth muscle cells (SMC) in synthetic cells. In primary cultures and subcultures before 10 and after 200 passages, SMC exhibit contractile-like, synthetic and transformed phenotypes, respectively, providing a good model for studying dedifferentiation process in vitro: the rationale for comparing these phenotypes of SMC in vivo rests in similar changes in cytoenzymatic and cytoskeletal features. In vivo, dedifferentiated SMC are transformed into foam cells by accumulating lipids. Thus, the aim of this study was to determine whether cholesterol metabolism undergoes changes in dedifferentiated cells and the three cultured phenotypes were compared in regard to their cholesterol efflux mechanisms. Phenotypic changes were shown to be associated with decrease in intracellular cholesterol apoprotein mediated efflux and translocation but also with decrease in high affinity binding sites for native HDL. Thus, the dedifferentiation process triggers a need for increased supply of cholesterol for membrane synthesis and efflux down-regulation mechanisms are aimed at maximizing cholesterol availability to the cell. Plasma membrane cholesterol efflux, which seems to be apoprotein-independent, decrease slightly with cell dedifferentiation suggesting either modifications in the dedifferentiated cell membranes physical properties. Taken together, these different results showed that dedifferentiation of arterial SMC is associated with decrease in the different steps of the efflux process, which could constitute one of the early events in their foam cell transformation.

Plasma factors affecting the in vitro conversion of high-density lipoproteins labeled with a non-transferable marker.

We studied the in vitro conversion of HDL3 labeled with a radioiodinated diacyl lipid associating peptide (diLAP). DiLAP was previously shown to be nontransferable, which permitted its' use as a reliable marker of HDL particles. DiLAP-labeled HDL3 was incubated for 23 h at 37 degrees C in human or rat plasma or in reconstituted media containing delipidated plasma and/or lipoproteins and/or partially purified CETP. At the end of the incubations, the samples were adjusted to a density of 1.125 g/ml and ultracentrifuged. The two resulting fractions containing HDL2 and HDL3, respectively, were analyzed by gradient gel electrophoresis. Depending upon experimental conditions, diLAP-labeled HDL3 was converted into HDL2b- and/or small HDL3c-like particles. LCAT inhibition and to a lesser extent CETP promoted the formation of small HDL3c. Reactivation of LCAT led to the disappearance of small HDL3c. No HDL3c formed from HDL2 even in the absence of LCAT activity. When the incubations were performed in the presence of 100 mM thimerosal, which inhibited PLTP but not CETP activity, the conversion of diLAP-labeled HDL3 into HDL2 was almost completely blocked. Collective consideration of these data indicates that the formation of small HDL is moderately facilitated by CETP; that small HDL are converted to larger HDL species by LCAT and that the transformation of HDL3 into HDL2 is a process which largely depends upon PLTP activity.

Alterations in composition and concentration of lipoproteins and elevated cholesteryl ester transfer in non-insulin-dependent diabetes mellitus (NIDDM).

Cholesteryl esters (CE) exchange between lipoproteins through the action of cholesteryl ester transfer protein (CETP). Situations at high risk for atherosclerosis are often accompanied by an accelerated net mass CE transfer (CET) from high density lipoproteins (HDL) to very low (VLDL) and low density lipoproteins (LDL). However, the question as to whether the net mass CET is increased or decreased in non-insulin-dependent diabetes mellitus (NIDDM) has led to controversial data. To clarify this point, we have undertaken a detailed study of CET in 105 NIDDM patients by comparison with 17 control subjects. Net mass CET was approximately doubled in NIDDM. Plasma CETP activity and unidirectional CET from HDL to VLDL + LDL (CETHDL-->VLDL + LDL) or from VLDL + LDL to HDL (CETVLDL + LDL-->HDL) were measured under controlled lipoprotein concentrations using radioisotopic assays. No difference was observed in plasma CETP activity between NIDDM and controls. In NIDDM, CETHDL-->VLDL + LDL and CETVLDL + LDL-->HDL were decreased by 25% and 20%, respectively, as a consequence of alterations in lipoprotein compositions. Net mass CET was highly correlated with plasma triglyceride (TG) concentration (r = 0.66, P < 0.001) but not with that of LDL-cholesterol (r = 0.06, P > 0.6). When TG levels were decreased following dietetic recommendations or insulinotherapy, the net mass CET was lowered accordingly. We conclude that net mass CET is accelerated in NIDDM in spite of a decreased unidirectional CETHDL-->VLDL + LDL. This results from a lowered CETVLDL + LDL-->HDL and from elevated TG concentration, and the latter probably reflects a concentration effect of VLDL.

Omega-3 fatty acids in smooth muscle cell phospholipids increase membrane cholesterol efflux.

The aim of our work was to determine whether fatty acid modifications in smooth muscle cell phospholipids affect cholesterol efflux and desorption. [3H]Cholesterol was used to label cholesterol pools in the whole cell or selectively in the plasma membrane. Cells were incubated for 12 h in order to increase oleate, linoleate, arachidonate, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in phospholipids. Cholesterol efflux was monitored using native or tetranitromethane modified high-density lipoprotein3 (HDL3). When all cholesterol pools were labeled, the efflux from cells treated with different fatty acids were not different. Plasma membrane cholesterol efflux remained unchanged after oleate, linoleate or arachidonate treatments, but was markedly increased after EPA and DHA enrichment, both with native HDL3 and with tetranitromethane-high-density lipoprotein. These results suggest that the positive effects of n-3 fatty acid consumption on the atherosclerotic process could be linked in part to an increase in plasma membrane cholesterol efflux from vascular smooth muscle cells.

High-density lipoprotein 3 stimulates phosphatidylcholine breakdown and sterol translocation in rat aortic smooth muscle cells by a phospholipase C/protein kinase C-dependent process.

The aim of this study was to elucidate signal transduction pathways following high-density lipoprotein 3 (HDL3) fixation to HDL high-affinity binding sites and leading to translocation of newly synthesized cholesterol to the plasma membrane pool for efflux. First, membrane phosphatidylcholine (PC) breakdown and 1,2-diacylglycerol (DAG) production were investigated following HDL3 or tetranitromethane (TNM)-HDL incubation with smooth muscle cells in culture. Second, newly synthesized cholesterol was labeled using [3H] mevalonolactone. Phospholipase C (PLC) and protein kinase C (PKC) were stimulated using carbachol and phorbol 12-myristate 13-acetate. Translocation and efflux of newly synthesized cholesterol were monitored using the cholesterol oxidase method and TNM-HDL as cholesterol acceptor. Results showed that: (1) native HDL3 but not modified HDL was able to stimulate PC breakdown and DAG formation; and (2) PLC and PKC stimulation using specific agents induce cholesterol translocation from intracellular to plasma membrane pool. Taken together, these two sets of results suggest that native HDL3 could induce cholesterol translocation by a PLC/PKC process in smooth muscle cells.

Cholesterol ester transfer and high-density lipoprotein conversion in normolipidemic, hypercholesterolemic, and hypertriglyceridemic non-insulin-dependent diabetics.

Non-insulin-dependent diabetes (NIDD) is a situation at elevated risk for atherosclerosis. The plasma concentration of high-density lipoprotein (HDL) is often lowered. This may be accompanied by an abnormal composition and profile of HDL subfractions. These abnormalities might result in part from a defect in the net cholesterol ester transfer (CET) from HDL to apo B-containing lipoproteins. In the present work, we have studied the net CET and HDL conversion in normolipidemic, hypercholesterolemic, and hypertriglyceridemic NIDD, by comparison with control subjects. HDL conversion was determined by gradient gel electrophoresis after 23 h incubation in plasma with HDL3 labeled with a nontransferable synthetic marker. The net CET in normolipidemic NIDD was similar to that of controls, while it was approximately doubled in hypercholesterolemic or hypertriglyceridemic NIDD. In all groups, HDL conversion was comparable, with the exception of hypertriglyceridemic NIDD. In the latter group, the labeled HDL2/HDL3 ratio was increased, indicating a more complete conversion that was correlated with the triglyceride/cholesterol ester ratio in HDL. In addition, when lecithin:cholesterol acyl transferase was inhibited, a distinct peak of small HDL particles appeared in the density range of HDL2 in contrast with the other groups where only small HDL3 was formed. Recombination experiments showed that these abnormalities were attributable to the plasma in which labeled HDL3 was incubated rather than to the origin (control or hypertriglyceridemic NIDD) of labeled HDL3. These data suggest that in NIDD, hypertriglyceridemia may result in abnormalities of HDL conversion due to alterations in HDL composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple abnormalities in the transfer of phospholipids from VLDL and LDL to HDL in non-insulin-dependent diabetes.

Transfers or exchanges of cholesterol esters and triglycerides between lipoproteins are mediated by a specialized protein referred to as cholesteryl ester transfer protein (CETP), whereas those of phospholipids (PLs) are facilitated by both CETP and a specific phospholipid transfer protein (PLTP). In the present study, the authors compared phospholipid transfer (PLT) in normal subjects and in patients with non-insulin-dependent diabetes (NIDD), which is associated with an increased risk of atherosclerosis. PLT was measured in different recombination experiments using an isotopic assay in which the transfer of labelled PLs from very low-density lipoprotein (VLDLs) and low-density lipoproteins (LDLs) to high-density lipoproteins (HDLs) was determined. This allowed discrimination between the roles of VLDLs + LDLs, HDLs, and plasma PLT activity (PLTA). VLDL + LDL-dependent PLT, HDL-dependent PLT and PLTA were decreased in NIDD. VLDL + LDL-dependent PLT was found to be negatively correlated with the PL/apolipoprotein B ratio, whereas HDL-dependent PLT was positively correlated with the HDL2/HDL3 and PL/apolipoprotein A-I ratios and negatively correlated with the flow activation energy at the HDL surface. The HDL2/HDL3 ratio was positively correlated with PLTA but not with CETP, which confirms previous reports suggesting that PLTP might act as an HDL conversion factor. These data show that several abnormalities in PLT occur in NIDD and raise the question as to whether a lowered PLT might be a new characteristic of dis factors associated with an increased risk of atherosclerosis.

High density lipoprotein interconversions in rat and man as assessed with a novel nontransferable apolipopeptide.

A nontransferable peptide analog of a plasma apolipoprotein diacyl lipid-associating peptide (diLAP) incorporates into model reassembled high density lipoproteins (R-HDL). In whole plasma in vitro, diLAP irreversibly transfers to native rat HDL2 and human HDL3, but not to rat HDL1 or human HDL2. The rate of transfer is dependent on the physical state of the lipid in the R-HDL. Exogenous cholesterol promotes the formation of larger HDL. When diLAP-labeled R-HDL were injected into rats, the diLAP that initially associated with HDL2 transferred to HDL1 over a period of 48 h. The rate of clearance of diLAP-labeled HDL was slower than that of apoA-I. The liver was the preferred site for diLAP-labeled HDL1 uptake. In contrast, diLAP-labeled HDL2 were associated with liver, ovaries, and adrenal glands, with the adrenal grands exhibiting the highest specific association. DiLAP was not found in the kidneys. These data show that 1) rat HDL is cleared more slowly than rat apoA-I; 2) HDL is removed from the plasma compartment as a particle; 3) there are tissue-specific differences in the removal of rat HDL1 and HDL2; 4) HDL2 is a precursor to HDL1; and 5) cholesterol and the activity of lecithin:cholesterol acyltransferase are essential to HDL remodeling.

VLDL-bound lipoprotein lipase facilitates the cholesteryl ester transfer protein-mediated transfer of cholesteryl esters from HDL to VLDL.

In recent years, it has been established that lipoprotein lipase (LPL) is partly associated with circulating lipoproteins. This report describes the effects of physiological amounts of very low density lipoprotein (VLDL)-bound LPL on the cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester transfer (CET) from high density lipoprotein (HDL) to VLDL. Three patients with severe LPL deficiency exhibited a strong decrease in net mass CET that was more than 80% lower than that of common hypertriglyceridemic subjects. Recombination experiments showed that this was due to an abnormal behavior of the VLDL fraction. Replacement of the latter by normal VLDL totally normalized net mass CET. We therefore prepared VLDL containing controlled amounts of bound LPL that we used as CE acceptors in experiments involving unidirectional radioisotopic CET measurements. These were carried out either in the absence or in the presence of inhibitors of LPL lipolytic activity. When LPL-induced lipolysis was totally blocked, the stimulating effect of the enzyme on the CETP-dependent CET was only reduced by about 50%, showing that it did not entirely result from its lipolytic action. These data were dependent upon neither the type of LPL inhibitor (E600 or THL) nor the source of CETP (delipidated plasma or partially purified CETP). Thus, in addition to the well-known stimulating effect of LPL-dependent lipolysis on CET, our work demonstrates that physiological amounts of VLDL-bound LPL may facilitate CET through a mechanism partially independent of its lipolytic activity.