Scavenger function of sinusoidal liver cells. Acetylated low-density lipoprotein is endocytosed via a route distinct from formaldehyde-treated serum albumin

Chemically modified proteins such as acetylated low-density lipoprotein (acetyl-LDL) and formaldehyde-treated serum albumin (f-Alb) infused intravenously are known to undergo receptor-mediated endocytosis by sinusoidal liver cells, major intravascular scavenger cells in vivo. The aim of the present study was to elucidate whether the endocytic uptake of acetyl-LDL and f-Alb is mediated by the same receptor or not. Experiments on the binding of 125I-acetyl-LDL to isolated rat liver sinusoidal cells revealed the presence of a specific, high-affinity, saturable, membrane-associated receptor with an apparent Kd = 7 micrograms of the ligand at 0 degrees C. Unlabeled acetyl-LDL effectively inhibited 125I-f-Alb binding to the cells. By contrast, the binding of 125I-acetyl-LDL to the cells was affected neither by unlabeled f-Alb nor by the antibody raised against the f-Alb receptor. These results indicate that the scavenger receptors for these two ligands are distinct from each other but similarly sensitive to polyanionic compounds.     

Receptor-mediated endocytosis of aldehyde-modified proteins by sinusoidal liver cells

Formaldehyde-treated serum albumin (f-Alb) is known to be endocytosed by sinusoidal lever cells via a receptor-mediated mechanism. The receptor purified from rat livers exhibited a molecular weight of 125,000, consisting of two glycoprotein components with molecular weights of 53,000 and 30,000, respectively. Experiments using antireceptor antibody demonstrated that the f-Alb receptor is distinct from the receptor that mediates endocytotic uptake of acetylated low-density lipoprotein, but they share a common property of being inhibited by several polyanions, suggesting that polyanion-sensitivity might play an important role in the scavenger function of simusoidal liver cells. Studies on the ligand specificity of this receptor revealed that a covalent modification by formaldehyde of a limited number of lysine residues in albumin has led to the formation of a receptor-recognition domain(s). Furthermore, in addition to formaldehyde, the ligand activity was also generated with albumin modified by other aliphatic aldehydes, such as glycoaldehyde and glyceraldehyde. This phenomenon was extended to several proteins other than albumin. These data suggest therefore that the f-Alb receptor originally described as being specific for albumin modified by formaldehyde may play a general role as a scavenger receptor for aldehyde-modified proteins.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Scavenger receptor for malondialdehyde-modified high density lipoprotein on rat sinusoidal liver cells

We report here the presence of a membrane-associated receptor which mediates endocytic uptake of malondialdehyde-modified high density lipoprotein (MDA-HDL) on sinusoidal liver cells. Binding of [125I]MDA-HDL to the cells was followed by internalization and degradation in lysosomes. The binding and lysosomal degradation of [125I]MDA-HDL were effectively inhibited by unlabeled MDA-HDL and acetyl-HDL. However, formaldehyde-treated serum albumin or low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by sinusoidal liver cells, did not affect the binding of [125I]MDA-HDL to the cells. These results indicate that a receptor for MDA-HDL is described as a distinct member among the scavenger receptors for chemically modified proteins.     

CD36-mediated endocytic uptake of advanced glycation end products (AGE) in mouse 3T3-L1 and human subcutaneous adipocytes

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. We here show that AGE-modified bovine serum albumin (BSA) is endocytosed by adipocytes via CD36. Upon differentiation, 3T3-L1 and human subcutaneous adipose cells showed marked increases in endocytic uptake and subsequent degradation of [(125)I]AGE-BSA, which were inhibited effectively by the anti-CD36 antibody. Ligand specificity of CD36 for modified BSAs was compared with that of LOX-1 and scavenger receptor class A. Effect of fucoidan on [(125)I]AGE-BSA binding showed a sharp contrast to that on [(125)I]-oxidized low density lipoprotein. These results implicate that CD36-mediated interaction of AGE-modified proteins with adipocytes might play a pathological role in obesity or insulin-resistance.     

Purification of a receptor for formaldehyde-treated serum albumin from rat liver

A membrane-associated receptor involved in a specific uptake of formaldehyde-treated serum albumin (f-Alb) was purified from rat livers by Triton X-100 solubilization of a 105,000 X g membrane preparation and affinity chromatography on an f-Alb-Sepharose column. The purified receptor exhibited Mr = 125,000, consisting of two noncovalently linked glycoprotein components with Mr = 53,000 and Mr = 30,000, respectively. Incubation of 125I-receptor with f-Alb, but not with native albumin, resulted in a marked shift of pI value from 5.9 to 5.1, reflecting the presence of a specific ligand-receptor interaction. The receptor incorporated into liposomes displayed a saturable binding to 125I-f-Alb and the binding was effectively replaced by the presence of unlabeled f-Alb, with binding parameters being similar to those obtained from 125I-f-Alb binding to the sinusoidal liver cell membrane (Horiuchi, S., Takata, K., and Morino, Y. (1985) J. Biol. Chem. 260, 475-481). Reaction of anti-f-Alb receptor antibody with extracts of sinusoidal cells resulted in a specific precipitation of two proteins whose molecular weights were identical to those for the purified receptor. The anti-receptor IgG fraction effectively blocked 125I-f-Alb binding to the sinusoidal cell membranes. These results indicate that the purified protein represents the membrane-associated receptor which is presumably involved in a specific uptake of this ligand from the circulation.     

A receptor for formaldehyde-treated serum albumin on human placental brush-border membrane

Formaldehyde-treated serum albumin (f-Alb) is known to be taken up and degraded by sinusoidal liver cells via receptor-mediated endocytosis. We report that 125I-labeled f-Alb (125I-f-Alb) binding to human placental brush-border membranes also occurs. This binding reached equilibrium within 40 min at 37 degrees C. Kinetic studies demonstrated the presence of saturable binding with an apparent Kd of 2.1 micrograms of f-Alb/ml and a maximal binding of 2.3 micrograms/mg of membrane protein at pH 7.5. Maximal binding was observed at between pH 7.5 and 8.0. 125I-f-Alb binding to the membranes was little inhibited by a 1000-fold molar excess of ovalbumin, human apo-transferrin and native bovine serum albumin. No binding was observed with membranes which had been pretreated with proteinase or trypsin. This f-Alb receptor was extremely heat-stable, since the binding was not abolished even by pretreatment of the membranes at 78 degrees C for 30 min. EDTA, Ca2+ and Mg/4 had no effect on 125I-f-Alb binding, so the binding was independent of divalent cations. These data suggest that a receptor specific for f-Alb exists on human placental brush-border membranes of syncytial trophoblasts.     

Association of advanced glycation end products with A549 cells, a human pulmonary epithelial cell line, is mediated by a receptor distinct from the scavenger receptor family and RAGE

Cellular interactions with advanced glycation end products (AGE)-modified proteins are known to induce several biological responses, not only endocytic uptake and degradation, but also the induction of cytokines and growth factors, combined responses that may be linked to the development of diabetic vascular complications. In this study we demonstrate that A549 cells, a human pulmonary epithelial cell line, possess a specific binding site for AGE-modified bovine serum albumin (AGE-BSA) (K(d) = 27.8 nM), and additionally for EN-RAGE (extracellular newly identified RAGE binding protein) (K(d) = 118 nM). Western blot and RT-PCR analysis showed that RAGE (receptor for AGE) is highly expressed on A549 cells, while the expression of other known AGE-receptors such as galectin-3 and SR-A (class A scavenger receptor), are below the level of detection. The binding of (125)I-AGE-BSA to these cells is inhibited by unlabeled AGE-BSA, but not by EN-RAGE. In contrast, the binding of (125)I-EN-RAGE is significantly inhibited by unlabeled EN-RAGE and soluble RAGE, but not by AGE-BSA. Our results indicate that A549 cells possess at least two binding sites, one specific for EN-RAGE and the other specific for AGE-BSA. The latter receptor on A549 cells is distinct from the scavenger receptor family and RAGE.     

FEEL-1 and FEEL-2 are endocytic receptors for advanced glycation end products

Advanced glycation end products (AGEs) are nonenzymatically glycosylated proteins, which accumulate in vascular tissues in aging and diabetes. Receptors for AGEs include scavenger receptors, which recognize acetylated low density lipoproteins (Ac-LDL) such as scavenger receptor class AI/AII (SR-A), cell surface glycoprotein CD36, scavenger receptor class B type I (SR-BI), and lectin-like oxidized low density lipoprotein receptor-1. The broad ligand repertoire of these receptors as well as the diversity of the receptors for AGEs have prompted us to examine whether AGEs are also recognized by the novel scavenger receptors, which we have recently isolated from a cDNA library prepared from human umbilical vein endothelial cells, such as the scavenger receptor expressed by endothelial cells-I (SREC-I); the fasciclin EGF-like, laminin-type EGF-like, and link domain-containing scavenger receptor-1 (FEEL-1); and its paralogous protein, FEEL-2. At 4 degrees C, (125)I-AGE-bovine serum albumin (BSA) exhibited high affinity specific binding to Chinese hamster ovary (CHO) cells overexpressing FEEL-1 (CHO-FEEL-1) and FEEL-2 (CHO-FEEL-2) with K(d) of 2.55 and 1.68 microg/ml, respectively, but not to CHO cells expressing SREC (CHO-SREC) and parent CHO cells. At 37 degrees C, (125)I-AGE-BSA was taken up and degraded by CHO-FEEL-1 and CHO-FEEL-2 cells but not by CHO-SREC and parent CHO cells. Thus, the ability to bind Ac-LDL is not necessarily a prerequisite to bind AGEs. The (125)I-AGE-BSA binding to CHO-FEEL-1 and CHO-FEEL-2 cells was effectively inhibited by Ac-LDL and polyanionic SR-A inhibitors such as fucoidan, polyinosinic acids, and dextran sulfate but not by native LDL, oxidized LDL, or HDL. FEEL-1, which is expressed by the liver and vascular tissues, may recognize AGEs, thereby contributing to the development of diabetic vascular complications and atherosclerosis.     

Endocytic uptake of nonenzymatically glycosylated proteins is mediated by a scavenger receptor for aldehyde-modified proteins

Long term incubation of proteins with glucose, named the Maillard reaction (Maillard, L. C. (1912) C. R. Acad. Sci. (Paris) 154, 66-68), gives rise to advanced glycosylation end product (AGE) with fluorescence, color, as well as cross-linked properties. The receptor-mediated endocytosis of AGE-proteins by macrophages was reported (Vlassara, H., Brownlee, M., and Cerami, A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5588-5592). The present study on the binding of AGE-bovine serum albumin (BSA) to rat peritoneal macrophages and sinusoidal liver cells demonstrated the presence of a saturable, high affinity receptor for AGE-BSA with Kd = 2.4 x 10(-7) M (macrophages) and 2.1 x 10(-7) M (sinusoidal cells). The cellular binding of AGE-BSA and its endocytic uptake by these cells were competitively inhibited by BSA preparations modified with aliphatic aldehydes such as formaldehyde or glycolaldehyde, ligands known to be specific for a scavenger receptor for aldehyde-modified proteins (Horiuchi, S., Murakami, M., Takata, K., and Morino, Y. (1986). J. Biol. Chem. 261, 4962-4966). These ligands also had a profound in vivo effect on the plasma clearance of 125I-AGE-BSA as well as its hepatic uptake. Thus, endocytic uptake of AGE-proteins by macrophages appeared to be mediated by a scavenger receptor for aldehyde-modified proteins. This provides evidence for the biological importance of the scavenger receptor in eliminating senescent macromolecules from the circulation.     

Scavenger receptors facilitate protein transport in the trophotaenial placenta of the goodeid fish,Ameca splendens (Teleostei: Atheriniformes)

In the goodeid placental analogue, trophotaeniae provide extraembryonic gut-derived exchange surfaces. Ameca splendens embryos possess endocytosing trophotaeniae that are capable of absorbing a dazzling array of proteinaceous substances. The iron core protein, native ferritin (NF), and several radioiodinated proteinaceous substances were used to study ligand and binding site pathways in the trophotaenial absorptive cells (TACs). Time sequence analysis of NF trafficking indicated an exclusively lysosomal pathway. Binding to TACs of NF was completely inhibitable by proteins containing multiple lysine residues such as apoferritin, bovine serum albumin (BSA), human transferrin (HTf), fetuin, hemoglobin, myoglobin, cytochrome c, ubiquitin, parvalbumin as well as the random copolymers, poly(Glu,Lys,Tyr)6:3:1 and poly(D-Glu,D-Lys)6:4. Peptide hormones and pepsin that contains only one lysine residue did not produce inhibitory effects. Radiolabels such as (125)I-BSA, (125)I-HTf and (125)I-poly(Glu,Lys,Tyr) bound to trophotaeniae in a specific saturable manner. Any two proteins were shown to hinder one another in getting hold of a binding site. Concentration-dependent (125)I-BSA binding and Scatchard analysis of the data revealed both low- and medium-affinity binding with apparent dissociation constants, K(d)s, of 3.4 x 10(-5) M and 2 x 10(-7) M, respectively. Binding of NF and radioiodinated proteins was inhibited in the presence of a large excess of L-Lys, D-Lys, and several dipeptides containing Lys. Both Ca(2+)-depletion and low pH dramatically reduced the TACs' capacity to bind proteins. The effects of acidotropic agents included a reversible loss of surface protein binding sites, tremendous vacuolation, and the arrest of lysosomal degradation. Collectively, present results demonstrate that TACs bind and absorb multiple proteinaceous substances through a mechanism satisfying the criteria of receptor-mediated endocytosis. It is concluded that scavenger protein binding sites are used to ingest proteins for lysosomal degradation, helping to meet the embryos' amino acid requirement.     

Identification of the receptor for atrial natriuretic factor on cultured vascular cells

Binding experiments with 125I-atrial natriuretic factor (ANF) followed by covalent attachment with disuccimidyl suberate show that the peptide binds predominantly to a protein of apparent molecular mass of 66,000 daltons on the cell surface of cultured bovine aortic smooth muscle cells. A minor protein species of 180,000 Mr is also visualized after cross-linking. Endothelial cells, however, whose ANF binding parameters differ substantially from smooth muscle cells, also appear to have qualitatively identical 125I-ANF binding proteins. The identity of these putative proteins, as the ANF receptor, is confirmed by findings that covalent attachment of 125I-ANF is saturable, concentration-dependent, and competed by nanomolar concentrations of unlabeled ANF. Furthermore, other peptide hormones such as angiotensin II, glucagon, or insulin are ineffective in competing for 125I-ANF binding and cross-linking to the receptor.     

Characterization of a membrane-associated receptor from rat sinusoidal liver cells that binds formaldehyde-treated serum albumin

When treated with formaldehyde, serum albumin is known to be taken up and degraded by sinusoidal liver cells via adsorptive endocytosis. The present study aimed at characterization and identification of the membrane-associated receptor on rat sinusoidal liver cells. Kinetic studies of binding of 125I-labeled formaldehyde-treated serum albumin (125I-f-Alb) with the membranes of sinusoidal liver cells demonstrated the presence of specific, high-affinity, saturable membrane-bound receptors with an apparent Kd = 8 micrograms of f-Alb/ml and the optimal pH at around 8.0. The 125I-f-Alb binding to the membranes was not inhibited by either native albumin, asialofetuin, methylamine-treated alpha 2-macroglobulin, mannan, or immune complexes. The binding process exhibited independence of calcium and susceptibility both to heat treatment and to destruction by proteases. The binding was inhibited by concanavalin A and the inhibition was effectively reversed by the presence of alpha-methyl-D-glucoside, a haptenic inhibitor for this lectin, indicating the glycoprotein nature of the receptor. The binding protein was extracted from the membrane preparations with octyl beta-D-glucopyranoside and immunoprecipitated by anti-ligand antibody as a complex with the ligand. Sodium dodecyl sulfate-gel electrophoresis of the immunoprecipitate revealed two polypeptide chains with molecular weights of approximately 53,000 and 30,000, respectively.     

The ligand activity of AGE-proteins to scavenger receptors is dependent on their rate of modification by AGEs

The cellular interaction of proteins modified with advanced glycation end-products (AGEs) is believed to induce several different biological responses, which are involved in the development of diabetic vascular complications. We report here that the ratio of protein glycation is implicated in its ligand activity to scavenger receptors. Although highly-modified AGE-bovine serum albumin (high-AGE-BSA) was significantly recognized by human monocyte-derived macrophages and Chinese hamster ovary cells which overexpress such scavenger receptors as CD36, SR-BI (scavenger receptor class B type-I), and LOX-1 (Lectin-like Ox-LDL receptor-1), the mildly-modified-AGE-BSA (mild-AGE-BSA) did not show any ligand activity to these cells. Furthermore, when (111)In-labeled high- or mild-AGE-BSA were injected into the tail vein of mice, the high-AGE-BSA was rapidly cleared from the circulation whereas the clearance rate of the mild-AGE-BSA was very slow, similar to the native BSA. These results demonstrate the first evidence that the ligand activity of the AGE-proteins to the scavenger receptors and its pharmacokinetic properties depend on their rate of modification by AGEs, and we should carefully prepare the AGE-proteins in vitro to clarify the physiological significance of the interaction between the AGE-receptors and AGE-proteins.     

Scavenger receptor of human monocytic leukemia cell line (THP-1) and murine macrophages for nonenzymatically glycosylated proteins

Long-term incubation of proteins with glucose undergo a series of nonenzymatic reactions to form advanced glycosylation end product (AGE) with fluorescence and brown color. The receptor for AGE-proteins was demonstrated in murine macrophages (Vlassara et al. (1985) Proc. Natl. Acad. Sci. USA 82. 5588). Our recent study with rat macrophages revealed that the receptor also recognized proteins modified with aliphatic aldehydes such as formaldehyde or glycolaldehyde, indicating its close identity to a scavenger receptor for aldehyde-modified proteins (Takata, K. et al. (1988) J. Biol. Chem. 263. 14819). This notion was tested in the present study with human monocytic leukemia cell line (THP-1 cells), human monocyte macrophages and murine peritoneal macrophages. Endocytic uptake of AGE-proteins and aldehyde-modified proteins was inhibited in a cross-competitive fashion. The receptor activities of THP-1 cells for AGE-albumin and aldehyde-modified proteins were induced synchronously by phorbol 12-myristate 13-acetate. Furthermore, upon reduction by NaBH4 of the Schiff base formed between proteins and glucose or aldehydes, no ligand activity was generated. However, once the ligand activity was generated, NaBH4 was no longer effective for the ligand activity. Thus, a structure in common between AGE-proteins and aldehyde-modified proteins may be crucial for recognition by the human macrophage receptor.     

CD36 is not involved in scavenger receptor-mediated endocytic uptake of glycolaldehyde- and methylglyoxal-modified proteins by liver endothelial cells

Circulating proteins modified by advanced glycation end-products (AGE) are mainly taken up by liver endothelial cells (LECs) via scavenger receptor-mediated endocytosis. Endocytic uptake of chemically modified proteins by macrophages and macrophage-derived cells is mediated by class A scavenger receptor (SR-A) and CD36. In a previous study using SR-A knockout mice, we demonstrated that SR-A is not involved in endocytic uptake of AGE proteins by LECs [Matsumoto et al. (2000) Biochem. J. 352, 233-240]. The present study was conducted to determine the contribution of CD36 to this process. Glycolaldehyde-modified BSA (GA-BSA) and methylglyoxal-modified BSA (MG-BSA) were used as AGE proteins. 125I-GA-BSA and 125I-MG-BSA underwent endocytic degradation by these cells at 37 degrees C, and this process was inhibited by several ligands for the scavenger receptors. However, this endocytic uptake of 125I-GA-BSA by LECs was not inhibited by a neutralizing anti-CD36 antibody. Similarly, hepatic uptake of (111)In-GA-BSA after its intravenous injection was not significantly attenuated by co-administration of the anti-CD36 antibody. These results clarify that CD36 does not play a significant role in elimination of GA-BSA and MG-BSA from the circulation, suggesting that the receptor involved in endocytic uptake of circulating AGE proteins by LEC is not SR-A or CD36.     

Competition of a growth stimulating-/cholecystokinin (CCK) releasing-peptide (monitor peptide) with epidermal growth factor for binding to 3T3 fibroblasts

The growth stimulating-/cholecystokinin (CCK) releasing-peptide (monitor peptide) is a peptide purified from rat bile-pancreatic juice on the basis of its stimulatory activity toward pancreatic enzyme secretion. Its multiple functions and peptide sequence suggested that it is distinct from epidermal growth factor (EGF). However, we found that the peptide competes with [125I]-EGF in the binding to Swiss 3T3 fibroblast cells to almost the same extent as unlabeled EGF does. [125I]-EGF binding was inhibited by 50% by the peptide at 82.8 ng/ml and by unlabeled EGF at 71.4 ng/ml. This suggests that the growth stimulating effect of the peptide on 3T3 fibroblasts is mediated via the EGF receptor, and also suggests that the partial homologous sequence between monitor peptide and EGF is required for the receptor binding, or that the EGF receptor has a broad ligand specificity.     

Identification of macrophage cell-surface binding sites for cationized bovine serum albumin.

Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens.     

Studies of the mechanism of the human platelet release reaction induced by immunologic stimuli. III. Relationship between the binding of soluble IgG aggregates to the Fc receptor and cell response in the presence and absence of plasma

Aggregated IgG coupled covalently with bis-diazobenzidine (BDB-IgG) and labeled with 3H-diazobenzene (3H-BDB-IgG) has been used to study the binding of soluble IgG aggregates to human platelets in relationship to the release of the contents of intracellular granules (e.g., serotonin). In washed cell suspensions a minimum of 0.14 to 0.2 mug 3H-BDB-IgG per 5 X 10(8) platelets (40 to 70 aggregates per cell) was required for the triggering of the release reaction and cell aggregation. Binding was independent of divalent cations. The Arrhenius plot gave a straight line between 0 to 37 degrees C and a Q10 of 1.6. Neither inhibitors of the release reaction nor energy metabolism, nor formaldehyde fixation of the platelets affected binding. Bound 3H-BDB-IgG was not significantly eluted by IgG, bovine albumin (BSA), buffer, or plasma. Binding to washed platelets was more strongly inhibited by human IgG than by F(ab')2, bovine IgG, human albumin (HSA), or BSA. Plasma was an even more effective inhibitor of both binding and release. Plasma deficient in IgG or depleted of complement retained its inhibitory capacity. In the presence of plasma, at physiologic ratios of plasma and platelets, no release of serotonin was observed. Binding, although inhibited in rate, nevertheless occurred. It was enhanced by divalent cation chelation and had a Q10 of 2.5. The release reaction of washed platelets to which 3H-BDB-IgG had been bound in the presence of HSA or BSA was also inhibited by the subsequent addition of plasma or plasma proteins (human IgG being more effective than bovine IgG, F(ab')2, HSA, or BSA). 3H-BDB-IgG bound in the presence of either plasma or human IgG did not induce release when the platelets were subsequently suspended in media lacking these proteins. Thus, it appears that the platelet Fc receptor binds 3H-BDB-IgG by a process which is effectively inhibited by plasma, or by free IgG with an intact Fc, and to some extent by high concentrations of other proteins. The effects of bound IgG aggregates are dependent on the other proteins present both during binding and subsequently added. The mechanism of such receptor modulation and its implications in vivo are discussed.     

Exogenous HSP70 becomes cell associated,but not internalized,by stressed arterial smooth muscle cells

Summary Cell death within atherosclerotic plaques leads to necrosis and rupture, resulting in vascular occlusion. We have previously demonstrated that addition of exogenous 70 kDa heat shock protein (HSP70) to arterial smooth muscle cells (aSMCs) in vitro can protect against toxins that may initiate necrosis. To determine whether exogenous HSP70 enters aSMCs or acts from outside cells to preserve viability, cultured rabbit aSMCs were stressed by serum deprivation and treated with fluorescently labeled (7-aminomethyl-4-coumarin-3-acetate) or¹²⁵I-radiolabeled HSP70. Cell-associated HSP70 was analyzed using Western blotting, fluoresence spectroscopy, and gamma counting/autoradiogarphy. Surface binding of HSP70 to aSMCs was differentiated from uptake by using trypsin treatment to degrade non-internalized HSP70. Specificity of HSP70 binding was tested by inhibiting uptake of¹²⁵I-HSP70 with excess unlabeled HSP70 or bovine serum albumin (BSA). The effect of unlabeled exogenous HSP70 on endogenous HSP synthesis was also tested. Exogenous HSP70 increased total cell-associated HSP70 2.9- to 3.6-fold over levels present in unstressed aSMCs. However, <5% of the exogenous HSP70 was trypsin-insensitive, indicating that bound HSP70 was not internalized. Binding of¹²⁵I-HSP70 was inhibited by both unlabeled HSP70 and BSA, implying a non-specific interaction with the plasmalemma. Exogenous HSP70 significantly lowered overall protein synthesis by serum-deprived aSMCs, but it did not specifically inhibit synthesis of endogenous HSPs after heat shock. The results indicate that exogenous HSP70 protects viability of stressed aSMCs through interactions with the cell surface rather than via internalization.     

Increased uptake of alpha-hydroxy aldehyde-modified low density lipoprotein by macrophage scavenger receptors

Reactive aldehydes can be formed during the oxidation of lipids, glucose, and amino acids and during the nonenzymatic glycation of proteins. Low density lipoprotein (LDL) modified with malondialdehyde are taken up by scavenger receptors on macrophages. In the current studies we determined whether alpha-hydroxy aldehydes also modify LDL to a form recognized by macrophage scavenger receptors. LDL modified by incubation with glycolaldehyde, glyceraldehyde, erythrose, arabinose, or glucose (alpha-hydroxy aldehydes that possess two, three, four, five, and six carbon atoms, respectively) exhibited decreased free amino groups and increased mobility on agarose gel electrophoresis. The lower the molecular weight of the aldehyde used for LDL modification, the more rapid and extensive was the derivatization of free amino groups. Approximately 50-75% of free lysine groups in LDL were modified after incubation with glyceraldehyde, glycolaldehyde, or erythrose for 24-48 h. Less extensive reductions in free amino groups were observed when LDL was incubated with arabinose or glucose, even at high concentration for up to 5 days. LDL modified with glycolaldehyde and glyceraldehyde labeled with (125)I was degraded more extensively by human monocyte-derived macrophages than was (125)I-labeled native LDL. Conversely, LDL modified with (125)I-labeled erythrose, arabinose, or glucose was degraded less rapidly than (125)I-labeled native LDL. Competition for the degradation of LDL modified with (125)I-labeled glyceraldehyde was nearly complete with acetyl-, glycolaldehyde-, and glyceraldehyde-modified LDL, fucoidin, and advanced glycation end product-modified bovine serum albumin, and absent with unlabeled native LDL.These results suggest that short-chain alpha-hydroxy aldehydes react with amino groups on LDL to yield moieties that are important determinants of recognition by macrophage scavenger receptors.