Degradation Of Acetylated LDL Research Articles

Suppression of viability and acetyl-LDL metabolism in RAW 264 macrophage-like and smooth muscle cells by bisphosphonates in vitro.

Etidronate and clodronate are bisphosphonates that inhibit the development of experimental atherosclerosis. Etidronate decreases the intimamedia thickness of carotid artery even in man. Liposome-encapsulated bisphosphonates inhibit the cellular metabolism of atherogenic, modified low-density lipoprotein (acetyl-LDL) by cultured macrophages. In the present study, the effects of new bisphosphonate tiludronate and nitrogen-containing bisphosphonate alendronate on cell viability and cellular uptake and degradation of acetyl-LDL were investigated in vitro with macrophages and arterial smooth muscle cells, which have a significant role in atherogenesis. Tiludronate and alendronate decreased the viability of RAW 264 macrophages at high concentration (1,000 microM; p < 0.05), while liposome-encapsulated drugs suppressed the viability at concentrations of 30-300 microM. At concentrations greater than or equal to 10 microM, tiludronate and alendronate inhibited the uptake and degradation of acetyl-LDL by RAW 264 cells in a concentration-dependent manner (p < 0.001). None of the bisphosphonates affected the viability of smooth muscle cells, and none but alendronate at a high concentration (1,000 microM) inhibited the uptake and degradation of acetyl-LDL by smooth muscle cells. The results show that tiludronate and alendronate inhibit the atherogenic activity of macrophages in vitro, as shown previously with etidronate and clodronate, providing further evidence for the antiatherogenic effects of bisphosphonates.

Endocytic uptake of advanced glycation end products by mouse liver sinusoidal endothelial cells is mediated by a scavenger receptor distinct from the macrophage scavenger receptor class A

Previous studies with peritoneal macrophages obtained from macrophage scavenger receptor class A (MSR-A) knock-out mice showed that the endocytic uptake of advanced glycation end products (AGE) by macrophages was mediated mainly by MSR-A. However, it is controversial whether the endocytic uptake of intravenously injected AGE proteins by liver sinusoidal endothelial cells (LECs) is similarly explained by receptor-mediated endocytosis via MSR-A. The present study was conducted to compare the capacity to endocytose AGE proteins in LECs and peritoneal macrophages obtained from MSR-A knock-out and littermate wild-type mice. The endocytic degradation capacity of MSR-A knock-out LECs for AGE–BSA was indistinguishable from that of wild-type LECs, whereas that of MSR-A knock-out peritoneal macrophages for AGE–BSA was decreased to 30% of that in wild-type cells. Similarly, the endocytic degradation of MSR-A knock-out LECs for acetylated low-density lipoprotein (acetyl-LDL) did not differ from that of wild-type LECs, whereas the endocytic degradation of acetyl-LDL by MSR-A knock-out peritoneal macrophages was less than 20% of that in wild-type cells. Furthermore, formaldehyde-treated serum albumin (f-Alb), a ligand known to undergo scavenger-receptor-mediated endocytosis by LECs, was effectively taken up by MSR-A knock-out LECs at a capacity that did not differ from that of wild-type LECs. Moreover, the endocytic uptake of AGE–BSA by LECs was effectively competed for by unlabelled f-Alb or acetyl-LDL. These results indicate that the scavenger-receptor ligands AGE proteins, acetyl-LDL and f-Alb are endocytosed by LECs through a non-MSR-A pathway.

Endocytic uptake of advanced glycation end products by mouse liver sinusoidal endothelial cells is mediated by a scavenger receptor distinct from the macrophage scavenger receptor class A

Previous studies with peritoneal macrophages obtained from macrophage scavenger receptor class A (MSR-A) knock-out mice showed that the endocytic uptake of advanced glycation end products (AGE) by macrophages was mediated mainly by MSR-A. However, it is controversial whether the endocytic uptake of intravenously injected AGE proteins by liver sinusoidal endothelial cells (LECs) is similarly explained by receptor-mediated endocytosis via MSR-A. The present study was conducted to compare the capacity to endocytose AGE proteins in LECs and peritoneal macrophages obtained from MSR-A knock-out and littermate wild-type mice. The endocytic degradation capacity of MSR-A knock-out LECs for AGE-BSA was indistinguishable from that of wild-type LECs, whereas that of MSR-A knock-out peritoneal macrophages for AGE-BSA was decreased to 30% of that in wild-type cells. Similarly, the endocytic degradation of MSR-A knock-out LECs for acetylated low-density lipoprotein (acetyl-LDL) did not differ from that of wild-type LECs, whereas the endocytic degradation of acetyl-LDL by MSR-A knock-out peritoneal macrophages was less than 20% of that in wild-type cells. Furthermore, formaldehyde-treated serum albumin (f-Alb), a ligand known to undergo scavenger-receptor-mediated endocytosis by LECs, was effectively taken up by MSR-A knock-out LECs at a capacity that did not differ from that of wild-type LECs. Moreover, the endocytic uptake of AGE-BSA by LECs was effectively competed for by unlabelled f-Alb or acetyl-LDL. These results indicate that the scavenger-receptor ligands AGE proteins, acetyl-LDL and f-Alb are endocytosed by LECs through a non-MSR-A pathway.

Macrophage specific overexpression of the human macrophage scavenger receptor in transgenic mice, using a 180-kb yeast artificial chromosome, leads to enhanced foam cell formation of isolated peritoneal macrophages

Macrophage scavenger receptors class A (MSR) are thought to play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins by macrophages in the vessel wall leading to foam cell formation. To investigate the in vivo role of the MSR in this process, a transgenic mouse model expressing both isoforms of the human MSR was generated. A 180-kb yeast artificial chromosome (YAC) containing the human MSR gene ( MSR1) with 60- and 40-kb flanking sequence at the 5′ and 3′ end, respectively, was obtained by reducing the size of a 1050-kb YAC by homologous recombination. This 180-kb YAC was microinjected into mouse oocytes. In the resulting transgenic mice, high levels of mRNA for both type I and type II human MSR1 were detected in peritoneal macrophages and trace levels in other organs, known to contain macrophage-derived cells. Using an antibody against the human MSR, the Kupffer cells in the liver were shown to contain the MSR protein. In vivo clearance of acetyl-LDL was not changed in the MSR1-transgenic mice. However, in vitro studies using peritoneal macrophages from the transgenic mice showed a two-fold increased degradation of acetyl-LDL and cholesterolester accumulation concomitant with a four-fold increase in foam cell formation, as compared to wild-type macrophages. Thus, macrophage specific overexpression of the MSR may lead to increased foam cell formation, which is one of the initial and crucial steps in atherogenesis.

Oxidative stress leads to cholesterol accumulation in vascular smooth muscle cells

The transformation of macrophages and smooth muscle cells into foam cells by modified low-density lipoproteins (LDL) is one of the key events of atherogenesis. Effects of free radicals have mainly been studied in LDL, and other than toxicity, data dealing with direct action of free radicals on cells are scarce. This study focused on the direct effects of free radicals on cholesterol metabolism of smooth muscle cells. A free radical generator, azobis-amidinopropane dihydrochloride, was used, and conditions for a standardized oxidative stress were set up in vascular smooth muscle cells. After free radical action, the cells presented an accumulation of cholesterol that appeared to be the result of: (i) an increase in cholesterol biosynthesis and esterification; (ii) a decrease in cell cholesteryl ester hydrolysis; and (iii) a reduced cholesterol efflux. All these parameters were opposed by antioxidants. In addition, oxidant stress induced an increased degradation of acetyl-LDL, whereas no change was noted for native LDL. From this data, it was concluded that cholesterol metabolism of vascular smooth muscle cells was markedly altered by in vitro treatment with free radicals, although cell viability was unaffected. The resulting disturbance in cholesterol metabolism favors accumulation of cholesterol and cholesteryl esters in vascular cells, and thus may contribute to the formation of smooth muscle foam cells.

Glycolaldehyde-modified low density lipoprotein leads macrophages to foam cells via the macrophage scavenger receptor.

It was shown that proteins modified with advanced glycation end products (AGE) are effectively endocytosed by macrophages or macrophage-derived cells in vitro, and immunohistochemical studies involving anti-AGE antibodies demonstrated the accumulation of AGE-modified proteins (AGE-proteins) in macrophage-derived foam cells in human atherosclerotic lesions in situ, suggesting the involvement of AGE-modified LDL in the atherogenic process in vivo. To examine this suggestion, LDL was modified with glycolaldehyde, a highly reactive intermediate of the Maillard reaction. Physicochemically, glycolaldehyde-modified LDL (GA-LDL) was characterized by increases in negative charge, fluorescence intensity, and reactivity to anti-AGE antibodies, properties highly similar to those of AGE-proteins. The cellular interaction of GA-LDL with mouse peritoneal macrophages showed that GA-LDL was specifically recognized and endocytosed, followed by lysosomal degradation. The endocytic uptake of GA-LDL by these cells was competitively inhibited by acetylated LDL (acetyl-LDL), and the endocytic degradation of acetyl-LDL was also competed for by GA-LDL. Furthermore, incubation of GA-LDL with these macrophages and Chinese hamster ovary cells overexpressing the macrophage scavenger receptor (MSR), but not with peritoneal macrophages from MSR-knockout mice, led to the intracellular accumulation of cholesteryl esters (CE). These results raised the possibility that AGE-modified LDL, if available in situ, is taken up by macrophages mainly via MSR and then contributes to foam cell formation in early atherosclerotic lesions.

Uptake and catabolism of modified LDL in scavenger-receptor class A type I/II knock-out mice.

The liver is the major organ responsible for the uptake of modified low-density lipoprotein (LDL) from the blood circulation, with endothelial and Kupffer cells as major cellular uptake sites. Scavenger-receptors, which include various classes, are held responsible for this uptake. Mice deficient in scavenger-receptor class A types I and II were created and the fate of acetylated LDL (Ac-LDL) in vivo and its interaction with liver endothelial, Kupffer and peritoneal macrophages was characterized. Surprisingly, the decay in vivo (t12 < 2 min), tissue distribution and liver uptake (at 5 min it was 77.4 +/- 4.6% of the injected dose) of Ac-LDL in the knock-out mice were not significantly different from control mice (t12 < 2 min and liver uptake 79.1 +/- 4.6% of the injected dose). A separation of mice liver cells into parenchymal, endothelial and Kupffer cells 10 min after injection of Ac-LDL indicated that in both control and knock-out mice the liver endothelial cells were responsible for more than 70% of the liver uptake. Both in control and knock-out mice, preinjection of polyinosinic acid (poly I, 200 microg) completely blocked the liver uptake, indicating that both in control and knock-out mice the scavenger-receptors are sensitive to poly I. Preinjection of suboptimal poly I concentrations (20 and 50 microg) provided evidence that the serum decay and liver uptake of Ac-LDL is more readily inhibited in the knock-out mice as compared with the control mice, indicating less efficient removal of Ac-LDL in vivo in the knock-out mice under these conditions. Studies in vitro with isolated liver endothelial and Kupffer cells from knock-out mice indicate that the cell association of Ac-LDL during 2 h at 37 degrees C is 50 and 53% of the control, respectively, whereas the degradation reaches values of 58 and 63%. For peritoneal macrophages from knock-out mice the cell association of Ac-LDL was identical to the control mice whereas the Ac-LDL degradation in cells from the knock-out mice was 17% of the control. The low degradation capacity of peritoneal macrophages from knock-out mice for Ac-LDL indicates that scavenger-receptor class A types I and II play a quantitative important role in the degradation of Ac-LDL by macrophages. In liver, the contribution of scavenger-receptor class A types I and II to the maximal uptake and degradation of Ac-LDL by endothelial and Kupffer cells was 40-50%. Binding studies performed at 4 degrees C indicate that the lower rates of degradation are due to a lower number of surface receptors on the cells from the knock-out mice. From the in vitro and in vivo data it can be concluded that in addition to the classic scavenger-receptors class A types I and II liver does contain additional novel poly I-sensitive scavenger-receptors that facilitate efficient removal of Ac-LDL from the blood circulation. The availability of the scavenger-receptor class A types I and II knock-out mice will stimulate further molecular identification of these receptors.

Acquisition of Secretion of Transforming Growth Factor-β1 Leads to Autonomous Suppression of Scavenger Receptor Activity in a Monocyte-Macrophage Cell Line, THP-1

Macrophage cells derived from the human monocytic leukemia cell line, THP-1, accumulate esterified cholesterol when cultivated in the presence of acetylated low density lipoprotein (Ac-LDL) through scavenger receptors (ScR). In the present study, we isolated a subtype of THP-1 cells that failed to accumulate esterified cholesterol when cultivated in the presence of Ac-LDL. The cells had negligible amounts of cell association and degradation of Ac-LDL compared with the parent THP-1 cells. The subtype THP-1 cells did not express ScR mRNA as well as that of lipoprotein lipase. In contrast, the expression of apolipoprotein E mRNA was greater than that found in parent THP-1 cells. The culture medium of subtype THP-1 cells treated with 12-O-tetradecanoylphorbol-13-acetate inhibited the uptake of Ac-LDL and the expression of ScR in parent THP-1 cells. After a 48-h incubation in the culture medium containing 12-O-tetradecanoylphorbol-13-acetate, the culture medium of differentiated subtype THP-1 cells contained 6.9 ng/ml transforming growth factor (TGF)-beta 1, while that of parent THP-1 cells secreted below detection level, which was less than 3 ng/ml. This inhibitory effect of the conditioned medium on the expression of ScR in parent THP-1 cells was abolished by pretreatment of the culture medium with anti-TGF-beta 1 antibodies. Parent THP-1 cells expressed as much TGF-beta 1 mRNA as sTHP-1 cells after stimulation of differentiation. Although the precursor forms of TGF-beta 1 that were synthesized in both parent and subtype THP-1 cells were of similar size and were expressed at similar levels, latent TGF-beta 1-binding protein, which is necessary for the secretion of TGF-beta 1, could only be co-immunoprecipitated with anti-TGF-beta 1 antibody from subtype THP-1 cells. This suggests that subtype THP-1 cells secrete TGF-beta 1 into the medium by forming a functional complex with the latent TGF-beta 1-binding protein. We conclude that subtype THP-1 cells could not take up Ac-LDL because ScR was inhibited (leading to a loss of function) caused by the secreted TGF-beta 1.

Binding and degradation of native and acetylated low density lipoproteins by monocyte derived macrophages of normal and hypercholesterolemic rabbits

Low density lipoprotein (LDL) was isolated from normal rabbits and was modified with acetic anhydride. Blood monocyte derived macrophages from normal and hypercholesterolemic (HC) rabbits were cultured, and on the 8th day the cells were incubated with native and modified LDL to study their binding and degradation. Macrophages from both normal and hypercholesterolemic rabbits express a limited number of receptors for normal plasma LDL whereas they exhibit a large number of receptors for acetyl LDL. There was no significant difference between binding and degradation of acetyl LDL by normal or hypercholesterolemic cells. However, binding and degradation of native LDL by monocytes of hypercholesterolemic cells were significantly less (p<0.0001) in comparison to binding and degradation of native LDL by normal cells indicating that there is a feedback inhibitory pathway in the cell that inhibits the synthesis of LDL receptors in the presence of excess LDL.

Expression Cloning of a Novel Scavenger Receptor from Human Endothelial Cells

Scavenger receptors mediate the endocytosis of chemically modified lipoproteins, such as acetylated low density lipoprotein (Ac-LDL) and oxidized LDL (Ox-LDL), and have been implicated in the pathogenesis of atherosclerosis. The evidence that endothelial cells possess scavenger receptor activity is substantial, and this property is widely used in the isolation of endothelial cells from vascular tissues. In the current study, we have isolated, by expression cloning, the cDNA encoding a novel type of scavenger receptor expressed by endothelial cells (SREC), which mediates the binding and degradation of Ac-LDL. The primary structure of the molecule has no significant homology to other types of scavenger receptors, including the recently cloned endothelial cell Ox-LDL receptor, a member of the C-type lectin family. The cDNA encodes a protein of 830 amino acids with a calculated molecular mass of 85, 735 Da (mature peptide). Chinese hamster ovary cells stably expressing SREC bound 125I-labeled Ac-LDL with high affinity (Kd = 3.0 microg/ml, approximately 1.7 nM) and degraded them via an endocytic pathway. Association of DiII-Ac-LDL were effectively inhibited by Ox-LDL, malondialdehyde-modified LDL, dextran sulfate, and polyinosinic acid, but not by natural LDL and heparin. The cloned receptor has several characteristic domain structures, including an N-terminal extracellular domain with five epidermal growth factor-like cysteine pattern signatures and an unusually long C-terminal cytoplasmic domain (391 amino acids) composed of a Ser/Pro-rich region followed by a Gly-rich region.

Inhibitory effects of HepG2 cell-derived apolipoprotein A-I-containing lipoproteins on cholesteryl ester accumulation in macrophages.

We investigated the mechanisms of inhibitory effects on foam cell formation of apolipoprotein A-I-containing lipoproteins secreted by HepG2 cells (HepG2-HDL) using mouse peritoneal macrophages. When macrophages were incubated with acetylated low-density lipoprotein (acetyl-LDL) in the presence of HepG2-HDL, cholesterol ester (CE) accumulation in cells was reduced by 63%. This inhibitory capacity was almost similar to that of plasma high-density lipoprotein (HDL). When macrophages were converted to foam cells with acetyl-LDL and then reacted with HepG2-HDL or plasma HDL, the HDL-induced CE reduction was 2.2-fold greater than HepG2-HDL. Similar results were obtained using apo E-free HepG2-HDL. Since the inhibitory effect of HDL on acetyl-LDL-induced CE accumulation in macrophages is due largely to its cholesterol efflux capacity, these results suggest the presence of an additional mechanism for the inhibition of CE accumulation by HepG2-HDL. To investigate the mechanism, acetyl-LDL was reisolated from HepG2-HDL by Sephacryl S-300 gel filtration after incubation in a cell-free system. Reisolated acetyl-LDL showed a significant reduction in electrophoretic mobility. The extent of CE accumulation by reisolated acetyl-LDL was reduced by 20% compared with control acetyl-LDL. Moreover, its endocytic degradation by macrophages was reduced by 28%. HepG2-HDL also inhibited macrophage degradation of acetyl-LDL as well as oxidized LDL, a likely atherogenic lipoprotein. This inhibitory effect was ascribed to the HepG2-HDL subfraction containing pre-beta HDL. Our results indicated that apo A-I-containing lipoproteins as a physiological model of nascent HDL may inhibit foam cell formation by reducing ligand activity of atherogenic lipoproteins. These data possibly suggest inhibitory function of nascent HDL for the formation of foam cells in vivo.

Dehydroepiandrosterone markedly inhibits the accumulation of cholesteryl ester in mouse macrophage J774-1 cells

To clarify the antiatherogenic mechanism of action of dehydroepiandrosterone (DHEA), we investigated the effects of DHEA on the accumulation of cholesteryl ester (CE) in cultured mouse macrophage J774-1 cells. The accumulation of CE in J774-1 cells in the presence of acetyl low density lipoprotein (AcLDL) and 10 −5 mol/1 DHEA was significantly reduced to 30% of the control values for 24 h. The marked effect of DHEA was observed as early as 6 h and continued at least for 48 h. This reduction by DHEA was dose-dependent and occurred starting at a DHEA dose of 5 × 10 −7 mol/1 for 24 h. DHEA treatment did not induced any changes in the cell surface binding, cell-association, or degradation of AcLDL. In comparison, the DHEA analogues, 8354 and 8356, which are known to be much stronger inhibitors of glucose 6-phosphate dehydrogenase than DHEA, did not show as marked an effect as DHEA on the accumulation of CE during the first 6 h. However, after 24–48 h of incubation, both 8354 and 8356 caused a marked reduction in the accumulation of CE similar to that observed with DHEA. A quantitative analysis of the cellular cholesterol content revealed that DHEA caused a marked reduction in CE with a concomitant continuous increase in free cholesterol (FC), while the DHEA analogues caused a marked reduction in CE with no change in FC. DHEA demonstrated little inhibitory effect on 25-hydroxycholesterol-driven esterification. Moreover, 10 −5 mol/1 DHEA induced a CE reduction in the foam cells induced by AcLDL. The CE-reducing capacity was also observed in the DHEA analogues. This CE-reducing capacity disappeared, however, when acyl CoA:cholesterol acyltransferase inhibitor, 58-035, was also present. Based on these findings, it can be concluded that the inhibitory effect of DHEA on the CE storage in response to AcLDL can be explained, at least in part, by two mechanisms. First, a recently published mechanism, namely, the inhibitory action of DHEA on lysosomal cholesterol transport, correlates well with the inhibition against foam cell transformation by DHEA in the early phase (at 6 h) observed in our study. With regard to the second mechanism, the CE-reducing capacity of DHEA from CE-laden foam cells, which appears to be related to a decreased cholesteryl ester cycle, may contribute to the inhibitory effect on the CE storage in the late phase (at 24 h and 48 h). These phase-specific inhibitory mechanisms of DHEA on the CE-storage may therefore partly explain the antiatherogenic action of DHEA.

Dehydroepiandrosterone markedly inhibits the accumulation of cholesteryl ester in mouse macrophage J774-1 cells

<h2>Abstract</h2> To clarify the antiatherogenic mechanism of action of dehydroepiandrosterone (DHEA), we investigated the effects of DHEA on the accumulation of cholesteryl ester (CE) in cultured mouse macrophage J774-1 cells. The accumulation of CE in J774-1 cells in the presence of acetyl low density lipoprotein (AcLDL) and 10⁻⁵ mol/1 DHEA was significantly reduced to 30% of the control values for 24 h. The marked effect of DHEA was observed as early as 6 h and continued at least for 48 h. This reduction by DHEA was dose-dependent and occurred starting at a DHEA dose of 5 × 10⁻⁷ mol/1 for 24 h. DHEA treatment did not induced any changes in the cell surface binding, cell-association, or degradation of AcLDL. In comparison, the DHEA analogues, 8354 and 8356, which are known to be much stronger inhibitors of glucose 6-phosphate dehydrogenase than DHEA, did not show as marked an effect as DHEA on the accumulation of CE during the first 6 h. However, after 24–48 h of incubation, both 8354 and 8356 caused a marked reduction in the accumulation of CE similar to that observed with DHEA. A quantitative analysis of the cellular cholesterol content revealed that DHEA caused a marked reduction in CE with a concomitant continuous increase in free cholesterol (FC), while the DHEA analogues caused a marked reduction in CE with no change in FC. DHEA demonstrated little inhibitory effect on 25-hydroxycholesterol-driven esterification. Moreover, 10⁻⁵ mol/1 DHEA induced a CE reduction in the foam cells induced by AcLDL. The CE-reducing capacity was also observed in the DHEA analogues. This CE-reducing capacity disappeared, however, when acyl CoA:cholesterol acyltransferase inhibitor, 58-035, was also present. Based on these findings, it can be concluded that the inhibitory effect of DHEA on the CE storage in response to AcLDL can be explained, at least in part, by two mechanisms. First, a recently published mechanism, namely, the inhibitory action of DHEA on lysosomal cholesterol transport, correlates well with the inhibition against foam cell transformation by DHEA in the early phase (at 6 h) observed in our study. With regard to the second mechanism, the CE-reducing capacity of DHEA from CE-laden foam cells, which appears to be related to a decreased cholesteryl ester cycle, may contribute to the inhibitory effect on the CE storage in the late phase (at 24 h and 48 h). These phase-specific inhibitory mechanisms of DHEA on the CE-storage may therefore partly explain the antiatherogenic action of DHEA.

Advanced glycation end products and their recognition by macrophage and macrophage-derived cells.

Modification of proteins by long-term incubation with glucose leads to the formation of advanced glycation end products (AGEs). AGE proteins are taken up by macrophages via the AGE receptor, which is similar to the macrophage scavenger receptor (MSR). In the present study, we compared the ligand specificity of the AGE receptor with that of MSR by three different experiments. The endocytic uptake of 125I-acetyl-LDL by RAW cells was effectively inhibited by unlabeled AGE-bovine serum albumin (BSA), whereas the inhibitory effect of acetyl-LDL on 125I-AGE-BSA was partial. Polyanions showing an effective inhibition for endocytic uptake of AGE-BSA were not always inhibitory for endocytic degradation of acetyl-LDL. These data, together with those obtained by three-dimensional fluorescence-activated cell sorter analysis, indicate that AGE proteins are recognized by more than two receptors, of which MSR is at least one. Finally, we examined whether MSR could mediate the endocytic uptake of AGE proteins by Chinese hamster ovary cells overexpressing bovine type II MSR (CHO-SRII cells). 125I-AGE-BSA underwent endocytic degradation by CHO-SRII cells, and this was effectively inhibited by unlabeled acetyl-LDL. These results clearly show that MSR mediates the endocytic uptake of AGE proteins, suggesting a new role of MSR in biological recognition of AGE in vivo.

Mechanism of Uptake of Copper-oxidized Low Density Lipoprotein in Macrophages Is Dependent on Its Extent of Oxidation

Several investigators have reported nonreciprocal cross-competition between unlabeled acetyl low density lipoprotein (LDL) and oxidized LDL for the degradation of the corresponding labeled LDLs. The failure of acetyl LDL to compete fully for the degradation of oxidized LDL has been interpreted as evidence for additional receptor(s) specific for oxidized LDL. In the present study, it is demonstrated that the ability of oxidized LDL to compete for the degradation of acetyl LDL is determined largely by its extent of oxidation. Extensively oxidized LDL competed for 90% of acetyl LDL degradation in murine macrophages, and hence there appears to be no pathway in these cells that is specific for acetyl LDL but not oxidized LDL. The reciprocal situation (competition by acetyl LDL for uptake and degradation of oxidized LDL) proved to be more complicated. Oxidized LDL is known to be susceptible to aggregation, and less than half of the aggregates found in the present experiments were large enough to be removed by filtration or centrifugation at 10,000 x g. When oxidized LDL was prepared under conditions that resulted in minimal aggregation, acetyl LDL competed for greater than 80% of oxidized LDL degradation. With more extensive oxidation and aggregation of LDL, acetyl LDL only competed for about 45% of oxidized LDL degradation, while polyinosinic acid remained an effective competitor. Individual preparations of oxidized LDL that differed in degree of oxidation were separated into aggregated and nonaggregated fractions, and it was shown that both fractions were competed to a similar degree by acetyl LDL in mouse peritoneal macrophages and in Chinese hamster ovary cells transfected with human scavenger receptor type I cDNA. Hence, aggregation by itself did not alter the apparent rate of uptake by the scavenger receptor pathway. These results indicate that the extent of oxidation of LDL affects its mechanism of uptake and that about half of the uptake of very extensively oxidized LDL appears to be via a pathway distinct from the scavenger receptor type I/II. The uptake of very extensively oxidized LDL was not affected by cytochalasin D, an inhibitor of phagocytosis. As well, it was not affected by an antibody to CD36 in human monocyte-derived macrophages or in THP-1 cells, suggesting that this alternate pathway does not involve CD36.

Modulation of macrophage scavenger receptor transport by protein phosphorylation

The identification of three highly conserved phosphorylation sites in the cytoplasmic domain of each of the monomeric subunits of the macrophage scavenger receptor suggests that protein phosphorylation may regulate this receptor pathway. To investigate this, mouse peritoneal macrophages were pretreated with either the protein phosphatase inhibitor okadaic acid or the protein kinase inhibitor staurosporine to modulate cellular protein phosphorylation and their effects on the metabolism of acetyl-LDL were measured. Both okadaic acid and staurosporine inhibited the degradation of acetyl-low density lipoprotein (LDL) without affecting cellular lactic dehydrogenase (LDH) levels. The inhibition by okadaic acid was due to a 70% decrease in acetyl-LDL binding whereas post-receptor processing was minimally affected. Calyculin A, another serine/threonine phosphatase inhibitor, also reduced acetyl-LDL binding, whereas lithium chloride, an inositol phosphatase inhibitor, did not. Okadaic acid did not decrease steady state receptor mRNA levels nor decrease the number of total cellular receptors, consistent with a posttranslational mechanism of action. Interestingly, protease sensitivity studies showed that the receptors were still located on the cell surface. These studies suggest that okadaic acid inhibits acetyl-LDL binding by causing the redistribution of surface receptors into a sequestered compartment or inactivating the receptors. In contrast, staurosporine produced a paradoxical increase in receptor expression (30%) but slowed post-receptor processing (2.3-fold decrease). The latter was due to an inhibition of ligand internalization (2.9-fold decrease) via a protein kinase C-independent mechanism. Macrophage pinocytosis was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also investigated and it was established that the receptor can be phosphorylated; however, changes in receptor function did not correlate with changes in the degree of receptor phosphorylation. Together these studies demonstrate that changes in cellular protein phosphorylation affect the expression, surface transport, and internalization of the macrophage scavenger receptor and suggest that the regulated phosphorylation/dephosphorylation of cellular proteins may be an important biochemical mechanism that controls normal processing of ligands by this receptor pathway.

Effect of 1,25-dihydroxyvitamin D3 on induction of scavenger receptor and differentiation of 12-O-tetradecanoylphorbol-13-acetate-treated THP-1 human monocyte like cells.

We investigated the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the expression of scavenger receptors in human monocytic cell line (THP-1 cells) treated for 24 h with 12-O-tetradecanoylphorbol-13-acetate (TPA) which induces their differentiation into macrophages. The capacity to degrade 125I-labeled acetyl low density lipoprotein (LDL) was developed in accordance with macrophage differentiation. The treatment with 10 nM 1,25(OH)2D3 for 72 h inhibited the degradation of acetyl LDL by THP-1 macrophages in a dose-dependent manner, suggesting that 1,25(OH)2D3 inhibits scavenging function in macrophages. In order to clarify the mechanism of its inhibitory effect on degradation of acetyl LDL, we performed the ligand binding assay using 125I-labeled acetyl LDL. Scatchard analysis revealed that 1,25(OH)2D3 decreased the number of scavenger receptors without changing the affinity for acetyl LDL. We next examined the effect of 1,25(OH)2D3 on the expression of scavenger receptor mRNA. The mRNA of type I scavenger receptor was first detected in THP-1 cells 4 days after the treatment with TPA, the mRNA level increased up to 6 days, and then decreased. The treatment with 1,25(OH)2D3 for 72 h dramatically decreased the mRNA levels after the acquisition of macrophage phenotypes as evidenced by nonspecific esterase staining. However, 1,25(OH)2D3 did not affect the activity of nonspecific esterase nor the induction of interleukin-1 beta mRNA by lipopolysaccharide in THP-1 macrophages. These findings suggest that 1,25(OH)2D3 exclusively decreases the expression of scavenger receptors in TPA-induced THP-1 macrophages without affecting the basic cellular functions as macrophages.

Isoprenoid requirement for acetyl LDL degradation and cholesterol accumulation in macrophages

Isoprenoid requirement for acetyl LDL degradation and cholesterol accumulation in macrophages

Age-Related Changes in the Metabolism of Acetylated Low-Density Lipoproteins by Peritoneal Macrophages From C57BL/6CrScl Mice

Macrophages are major precursors of foam cells in atherosclerotic lesions. Acetylated low-density lipoproteins (acetyl LDL) taken up by macrophages through scavenger receptors are degraded by lysosomes and the released cholesterol is re-esterified, leading to foam cell formation. The ability of resident peritoneal macrophages from C57BL/6CrScl mice to form foam cells in relation to the donor age was assessed by the cholesterol esterification and the metabolism of acetyl LDL. The incorporation of 14C-oleate (complexed to albumin) into cellular cholesteryl esters in the presence of acetyl LDL (100 micrograms/ml) was significantly greater in macrophages from senescent mice (24-25 months) than in cells from young (3-4 months) mice (p < .001). The degradation and cellular association of acetyl LDL by macrophages from senescent mice were significantly greater than macrophages from mature mice, (p < .001 and p < .01, respectively), whereas the binding of acetyl LDL was similar in peritoneal macrophages from mature and senescent mice. These results suggest that the uptake and degradation of acetyl LDL, and the re-esterification by macrophages increase with advancing age and that the ability of macrophages to form foam cells increases with aging. The enhanced ability of senescent macrophages to form foam cells might contribute to the development and progression of atherosclerosis related to the aging process.

Characterization of the scavenger receptor on bovine cerebral endothelial cells in vitro.

Primary cultures of bovine brain capillary endothelial cells (BCEC), possessing tight junctions and high levels of gamma-glutamyl transpeptidase, were used as an in vitro model for the blood-brain barrier. The interaction of acetylated low density lipoprotein (AcLDL) with BCEC was studied to characterize the scavenger receptor on these cells. A saturable high affinity binding site was found with a dissociation constant of AcLDL of 5.4 micrograms/ml (3.1 nM) and a maximal binding ranging from 284 to 626 ng of AcLDL/mg of cell protein for eight primary cultures, and independent of the presence of calcium. Cell association was coupled to degradation, and both could be effectively competed for by polyinosinic acid and AcLDL but not by low density lipoprotein or by high density lipoprotein. Prolonged incubation showed an accumulation of the ligand in the cells. The rate of degradation of AcLDL was approximately 10-20-fold lower in BECEC than that of peripheral endothelial cells. No evidence for lysosomal degradation could be obtained. Binding of 1,1'-dioctadecyl-3,3,3'-tetramethylindocarboxyamine perchlorate-labeled AcLDL by BCEC was observed, which could be competed for by an excess of unlabeled AcLDL and polyinosinic acid. We have shown that in vitro BCEC possesses specific binding sites for AcLDL, whereas these cells show a relatively low degradative capacity.