Role of macrophage-expressed adipocyte fatty acid binding protein in the development of accelerated atherosclerosis in hypercholesterolemic mice.

Atherosclerosis is an inflammatory disease process associated with elevated levels of plasma cholesterol, especially low-density lipoproteins. The latter become trapped within the arterial wall and are oxidized and taken up by macrophages to form foam cells. This process is an initiating event for atherosclerosis. Fatty acid binding proteins (FABP) are involved in fatty acid metabolism and cellular lipid transport, and adipocyte FABP (aP2) is also expressed in macrophages. We recently generated mice lacking both apolipoprotein (Apo)E and aP2 (ApoE-/-aP2-/-) and found that these mice, compared with ApoE-/- mice, developed markedly smaller atherosclerotic lesions that contained fewer macrophages. Here we investigated the mechanism(s) responsible for this prevention of atherosclerotic lesion formation. Bone marrow transplantations were performed in ApoE-/- mice, receiving cells from either ApoE-/- or ApoE-/-aP2-/- mice. The lack of aP2 in donor marrow cells led to the development of smaller (5.5-fold) atherosclerotic lesions in the recipient mice. No differences were found in plasma cholesterol, glucose, or insulin levels between recipients of bone marrow cells from ApoE-/- or ApoE-/-aP2-/- mice. However, the expression of chemoattractant and inflammatory cytokines was decreased in macrophages from ApoE-/-aP2-/- mice compared with ApoE-/- mice, which may contribute to the decrease in atherosclerotic lesion formation. Taken together, we demonstrate the importance of macrophage aP2 in the development of atherosclerotic lesions.

beta-Migrating very low density lipoprotein (beta VLDL) activates smooth muscle cell mitogen-activated protein (MAP) kinase via G protein-coupled receptor-mediated transactivation of the epidermal growth factor (EGF) receptor: effect of MAP kinase activation on beta VLDL plus EGF-induced cell proliferation.

This study examined the premise that the atherogenic lipoprotein, beta-migrating very low density lipoprotein (betaVLDL), might activate the mitogen-activated protein (MAP) kinases ERK1/ERK2, thereby contributing to the induction of smooth muscle cell proliferation in atherosclerosis. The data show that betaVLDL activates rabbit smooth muscle cell ERK1/ERK2. Interestingly, ERK1/ERK2 activation is mediated by G protein-coupled receptors that transactivate the epidermal growth factor (EGF) receptor. betaVLDL-induced MAP kinase activation depends on Ras and Src activity as well as protein kinase C. The inhibition of lysosomal degradation of betaVLDL has no effect on ERK1/ERK2 activation. The contribution of betaVLDL-induced activation of ERK1/ERK2 to smooth muscle cell proliferation was also explored. betaVLDL induces expression of egr-1 and c-fos mRNA. Despite its ability to stimulate early gene expression, betaVLDL alone is unable to inspire quiescent cells into S phase. When added in conjunction with EGF, however, stimulation of [(3)H]thymidine incorporation into DNA and an increase in histone gene expression are observed. Moreover, betaVLDL plus EGF synergistically induce cyclin D1 expression and down-regulate p27(KIP1) expression. The addition of either betaVLDL or EGF stimulates a robust activation of ERK1/ERK2, but the addition of both agents simultaneously sustains the activation for a longer time period. Inhibition of MAP kinase kinase, pertussis toxin-sensitive G proteins, the EGF receptor, or protein kinase C blocks betaVLDL plus EGF-induced proliferation, demonstrating that activation of the betaVLDL-induced signaling pathway results in smooth muscle cell proliferation.

Serum amyloid A in Alzheimer's disease brain is predominantly localized to myelin sheaths and axonal membrane.

Immunohistochemical localization of the injury specific apolipoprotein, acute phase serum amyloid A (A-apoSAA), was compared in brains of patients with neuropathologically confirmed Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD); Pick's disease (Pick's), dementia with Lewy bodies (DLB), coronary artery disease (CAD), and schizophrenia. Affected regions of both AD and MS brains showed intense staining for A-apoSAA in comparison to an unaffected region and non-AD/MS brains. The major site of A-apoSAA staining in both diseases was the myelin sheaths of axons in layers V and VI of affected cortex. A-apoSAA contains a cholesterol binding site near its amino terminus and is likely to have a high affinity for cholesterol-rich myelin. These findings, along with our recent evidence that A-apoSAA can inhibit lipid synthesis in vascular smooth muscle cells suggest that A-apoSAA plays a role in the neuronal loss and white matter damage occurring in AD and MS.

Apolipoprotein serum amyloid A down-regulates smooth-muscle cell lipid biosynthesis.

The addition of acute-phase apolipoprotein serum amyloid A (SAA) to cultured aortic smooth-muscle cells caused a decrease in the incorporation of [(14)C]acetate into lipids. Optimal inhibition of lipid biosynthesis was achieved with 2 microM SAA, and the effect was maintained for up to 1 week when SAA was included in the culture medium. Lipid extracts were subjected to TLC and it was determined that the SAA-induced decrease in [(14)C]acetate incorporation into lipids was attributable to decreases in cholesterol, phospholipid and triglyceride levels. The accumulated mass of cholesterol and phospholipid in SAA-treated cultures was significantly less than that of controls, with no change in the accumulated protein. Moreover, SAA had no effect on either protein synthesis or DNA synthesis, suggesting that SAA specifically alters lipid synthesis. By using a peptide corresponding to the cholesterol-binding domain of acute-phase SAA (amino acids 1-18), it was shown that this region of the molecule was as effective as the full-length protein in decreasing lipid synthesis and the accumulation of cholesterol and phospholipid. The implications of these findings for atherosclerosis and Alzheimer's disease are discussed.

The role of the carboxy terminus of tropoelastin in its assembly into the elastic fiber.

Tropoelastin, the soluble precursor protein of insoluble amorphous elastin, contains repeating segments that are important for the characteristic elasticity and crosslinking sites of mature elastin. In addition, there is a unique carboxy terminal domain that is encoded by exon 36 of the elastin gene, and it has been suggested that this region may play a role in the process of insolubilization. The contribution of exon 36 to the maturation of tropoelastin into insoluble elastin was probed in these studies. Neonatal rat aortic smooth muscle cells were cultured and the fate of [3H] Lys labeled human recombinant tropoelastin (hrTE) molecules added to the cultures was monitored. In comparison to the hrTE containing the region encoded by exon 36, hrTE molecules lacking this domain were less efficiently incorporated into elastin, as evidenced by a decrease in NaOH insoluble radioactivity. Specific residues within the domain encoded by exon 36 were targeted for further study in experiments in which the two Cys residues were reduced and alkylated, and/or the four basic Arg-Lys-Arg-Lys residues at the carboxy terminus were removed. Both of these modifications resulted in decreased incorporation into elastin equivalent to the complete removal of the carboxy terminus. Prior treatment of the cell layer with elastase reduced the efficiency of insolubilization of hrTE containing the domain encoded by exon 36, but had no effect on the processing of molecules lacking this region. These data suggest that exon 36 of the elastin gene contributes to normal efficient incorporation of tropoelastin into the elastin fiber.

Retinal Muller glia secrete apolipoproteins E and J which are efficiently assembled into lipoprotein particles.

We have shown that apolipoprotein E (ApoE) is synthesized by Muller cells, the major glial cell of the rabbit retina, and secreted into the vitreous after which it is taken up by retinal ganglion cells and rapidly transported into the optic nerve [Amaratunga et al., J. Biol. Chem. 271 (1996) 5628-5632]. In this report we demonstrate that the ApoE secreted by Muller cells in vivo and in culture is efficiently assembled into lipoprotein particles. Apolipoprotein J (ApoJ) is also synthesized by these cells and assembled into lipoprotein particles. The lipoproteins are triglyceride-rich and contain cholesterol esters and free cholesterol. They are heterogeneous, with densities between 1.006 and 1.18 and diameters between 14 and 45 nm. We discuss the possible role of these lipoproteins in supplying the needs of neurons for lipids, especially long axonal projection neurons such as retinal ganglion cells, which are vulnerable to age-related neurodegenerative diseases including Alzheimer's disease.

Regulation of extrahepatic apolipoprotein serum amyloid A (ApoSAA) gene expression by interleukin-1 alpha alone: synthesis and secretion of ApoSAA by cultured aortic smooth muscle cells.

Serum amyloid A apolipoproteins (apoSAA) appear to compromise the ability of high density lipoprotein to protect against atherosclerosis and it is of interest to determine whether aortic smooth muscle cells can contribute to local pools of apoSAA in the presence of cytokines that are known to stimulate acute phase apoSAA (A-apoSAA) synthesis in the liver. In this study, the regulation of A-apoSAA synthesis was monitored in cultured neonatal rabbit aortic smooth muscle cells. Constitutive apoSAA3 gene expression was minimal, and only detectable by amplification of the mRNA by reverse transcriptase-polymerase chain reaction. ApoSAA3 gene expression and protein synthesis were stimulated by IL-1 alpha; as little as 0.01 ng/ml of IL-1 alpha stimulated an increase in steady state levels of apoSAA3 mRNA. Interestingly, IL-6 (which is required in addition to IL-1 alpha for the optimal synthesis of A-apoSAA by human hepatoma cells) had little if any effect on apoSAA3 synthesis by the smooth muscle cells. In a time course, it was shown that the stimulation of apoSAA3 mRNA levels was apparent by 1-2 h after the addition of cytokine, and that levels remained elevated in the presence of the cytokine for at least 48 h. Immunoprecipitation using an antiserum directed against apoSAA3 revealed that IL-1 alpha stimulated the synthesis and secretion of apoSAA3 protein in a manner that was consistent with apoSAA3 mRNA expression. The implications of these findings in atherogenesis are discussed.

Amino terminal region of acute phase, but not constitutive, serum amyloid A (apoSAA) specifically binds and transports cholesterol into aortic smooth muscle and HepG2 cells.

The human apoSAA proteins comprise both acute phase (apoSAA1, apoSAA2) and constitutive (apoSAA4) isoforms; all are expressed in human atherosclerotic lesions as well as in liver. Recombinant acute phase apoSAA binds cholesterol with an affinity of approximately 170 nM and enhances cholesterol uptake by HepG2 cells (J. Lipid Res. 1995. 36:37-46). In the present study, we sought to define the region of acute phase apoSAA involved in cholesterol binding and to investigate the ability of constitutive apoSAA4 to bind cholesterol. Binding of [3H]cholesterol to apoSAAp was inhibited by unlabeled cholesterol (1-100 nM), but not significantly by vitamin D and estradiol. Direct binding of acute phase, but not constitutive, apoSAA to the surfaces of polystyrene microtiter wells was strongly diminished in the presence of cholesterol. The ability of apoSAAp to bind cholesterol was inhibited by antibodies to human apoSAA1 and to peptide 1-18 of apoSAA1. There was only slight inhibition of cholesterol binding by antibodies to peptide 40-63, and no inhibition by antibodies to peptides spanning regions containing amino acid residues 14-44 and 59-104. [3H]cholesterol uptake by neonatal rabbit aortic smooth muscle and HepG2 cells was enhanced by a synthetic peptide corresponding to amino acids 1-18 of hSAA1, but not by peptides corresponding to amino acids 1-18 of hSAA4. [3H]cholesterol uptake by HepG2 cells was slightly increased by a peptide corresponding to amino acids 40-63 of hSAA1. These findings suggest that apoSAA modulates the local flux of cholesterol between cells and lipoproteins during inflammation and atherosclerosis.

Apolipoprotein E is synthesized in the retina by Müller glial cells, secreted into the vitreous, and rapidly transported into the optic nerve by retinal ganglion cells.

We have investigated the synthesis and transport of apoE, the major apolipoprotein of the central nervous system, in the retina of the living rabbit. Four hours after the injection of [35S]methionine/cysteine into the vitreous, 44% of [35S]Met/Cys-labeled apoE is in soluble and membrane-enclosed retinal fractions, while 50% is in the vitreous. A significant amount of intact [35S]Met/Cys-labeled apoE is rapidly transported into the optic nerve and its terminals in the lateral geniculate and superior colliculus within 3-6 h in two distinguishable vesicular compartments. Müller glia in cell culture also synthesize and secrete apoE. Taken together, these results suggest that apoE is synthesized by Müller glia and secreted into the vitreous. ApoE is also internalized by retinal ganglion cells and/or synthesized by these cells and rapidly transported into the optic nerve and brain as an intact molecule. We discuss the possible roles of retinal apoE in neuronal dynamics.

Apolipoprotein E expression in aortic smooth muscle cells: the effect of beta VLDL.

The expression of apolipoprotein E in cultured neonatal rabbit aortic smooth muscle cells was examined. Northern blot analysis determined that there was a single RNA transcript of approximately 1.2 kb. Moreover, a polyclonal antibody against rabbit apolipoprotein E was prepared in a goat and used in immunoprecipitations to demonstrate that the cultured cells secreted apolipoprotein E into the media. A double band typical of apolipoprotein E migrated at apparent molecular masses of 37 and 39 kDa. Analysis of steady-state levels of apolipoprotein E mRNA demonstrated that expression increased as cell seeding density increased. When examined as a function of time in culture, there were two peaks of expression evident 1 day and 8 days after seeding. The addition of beta VLDL (beta-very low density lipoprotein) to smooth muscle cells increased both [3H]thymidine incorporation into DNA as well as cell number and these increases were accompanied by a decrease in the levels of apolipoprotein E mRNA in cells treated with the lipoprotein for 1 and 7 days. After incubation of the cultures with beta VLDL for 1 week, the cells were radiolabeled with [35S]methionine and the media was subjected to immunoprecipitation with anti-apolipoprotein E. The data revealed that the amount of apolipoprotein E secreted into the media decreased in the presence of beta VLDL. In summary, these results show that apolipoprotein E expression in aortic smooth muscle cells is regulated by cell density, time in culture, cell proliferative state, and beta VLDL addition. These observations may have relevance to the conditions that are known to accompany the development of the atherosclerotic lesion.

Superoxide production by macrophages stimulated in vivo with synthetic ether lipids.

The anticancer activity of synthetic ether lipids may depend in part upon their ability to activate cells of the monocyte/macrophage lineage. In the present study, we have sought to determine whether 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and related ether lipids enhance superoxide production by mouse peritoneal macrophages. Ether lipids were administered intraperitoneally to C57BL/6 mice 4 d after injection with thioglycollate broth. Elicited peritoneal macrophages were harvested and purified one day later, and superoxide production was detected by measuring the superoxide dismutase inhibitable reduction of cytochrome c. Low levels of superoxide were secreted by macrophages in the absence of phorbol 12-myristate 13-acetate (PMA). When PMA was added in vitro to macrophages from ET-18-OMe-treated mice, these cells secreted 194.2 nmol superoxide/mg protein in comparison to 53.5 nmol superoxide/mg protein for PMA-treated control cells. The in vitro treatment of the macrophages with ET-18-OMe was not effective in stimulating superoxide secretion. Macrophages harvested from mice treated with a series of ether lipids (with and without phosphorus) were examined, and superoxide secretion was found to vary with structure. AM-18-OEt and CP-7 were the most effective compounds, secreting 8.6 and 11.9 times more superoxide, respectively, than PMA-stimulated control cells. Moreover, direct cytotoxicity of the compounds for HL60 human promyelocytic leukemic cells did not necessarily correlate with the ability of each drug to increase macrophage superoxide production.

Long-term treatment of neonatal aortic smooth muscle cells with beta VLDL induces cholesterol accumulation.

A model for smooth muscle derived foam cells was developed by treating smooth muscle cells isolated from the aortae of neonatal rabbits with beta VLDL for up to 1 month. Hyperlipidemic beta VLDL isolated from cholesterol fed rabbits induced proliferation of the cells that were maintained in lipid deficient serum. In addition, the lipoprotein fraction stimulated [14C]oleic acid incorporation into [14C]cholesteryl ester, even in cultures that had been chronically exposed to the lipoprotein. The accumulation of cholesterol was evaluated and small amounts of cholesteryl ester were demonstrated in cultures treated for 3 days with beta VLDL. However, continued exposure to the lipoprotein resulted in larger elevations in total cholesterol, approximately 65% of which was in the esterified form in cultures treated with 100 micrograms beta VLDL/ml for 24 days. When cholesterol levels were examined as a function of time, it was determined that both total cholesterol and cholesteryl ester levels increased. Approximately 2-3 weeks after lipoprotein was introduced to the culture, maximum levels were attained. Triglyceride levels were also measured and found to increase more than two-fold in cultures that had been incubated in the presence of beta VLDL for 24 days, when compared to cultures incubated in its absence. Examination of the cultures by electron microscopy revealed intracytoplasmic lipid droplets in beta VLDL treated cells. These results suggest that beta VLDL treatment of neonatal aortic smooth muscle cells provides an ideal model in which to study the lipid laden smooth muscle cells that characterize the atherosclerotic plaque.

Alterations of tropoelastin biosynthesis by elastase damage to smooth muscle cell matrices.

The effect of porcine pancreatic elastase (PPE)-induced proteolysis of the extracellular matrix on elastin biosynthesis in neonatal rat aortic smooth muscle cell cultures (NRSMC) was examined. The quantity of insoluble elastin remaining in the damaged cultures decreased with increasing amounts of enzyme used, however no significant cell damage was demonstrated. The accumulation of soluble elastin (tropoelastin) was examined in enzyme injured and control cultures by radiolabelling with [3H]-valine for 4 hours. The tropoelastin content of both the cell layer and media were less in injured cultures on the day of injury and up to one week later when compared to control cultures. In addition, experiments in which cultures were radiolabelled for 15 minutes demonstrated that the biosynthesis of tropoelastin was decreased in the enzyme treated cultures. Moreover, the incorporation of radiolabelled elastin into the insoluble matrix also decreased. Steady-state levels of elastin mRNA showed no differences between injured and control cultures, which suggested that elastin synthesis is affected at a translational or post-translational level.

A controlled precursor add-back model of elastogenesis in smooth muscle cell cultures.

Neonatal rat aortic smooth muscle cell cultures are capable of synthesizing and accumulating relatively large amounts of insoluble elastin in the extracellular matrix. There are two major soluble elastin molecules in these cultures, one of 77 kDa (protropoelastin) and the other of 71 kDa (tropoelastin). We examined the ability of the cell culture system to insolubilize exogenously added soluble elastin precursor moieties into the elastin matrix. To accomplish this, cultures were allowed to develop an enriched elastic fiber matrix for approximately two weeks in first passage. This accumulated matrix then served as the "substrate" for the exogenously added precursor elastin molecules. Culture-derived radioactive soluble elastin was added to the "substrate" cultures and the presence of radioactivity in the insoluble elastin as well as in the lysine-derived crosslinks unique to elastin (desmosines) was measured. When purified [3H]-valine radiolabeled protropoelastin was used, more than 15% of the radioactivity added was detected in the alkali-resistant insoluble elastin within 24 hours. After an initial 4-hour incubation of the cells with [3H]-lysine-labelled soluble elastin, most of the radioactivity in the insoluble elastin was associated with the lysine and only a negligible amount was detected in the desmosines. However, during a 16-day chase period, the ratio of radioactive desmosines to lysine increased dramatically, suggesting that not only insolubilization, but crosslinking occurs as well. The add-back system described herein should provide a means to probe the molecular properties of protropoelastin and increase our understanding of the mechanisms of elastic fiber formation.

beta-VLDL-induced alterations in growth potentiating activity produced by mononuclear phagocytes.

This report describes the enhancement of growth potentiating activity produced by mononuclear phagocytes that were incubated with beta-migrating very low density lipoproteins (beta-VLDL). Conditioned media harvested from cultured human peripheral blood monocytes incubated in the presence or absence of the lipoprotein were evaluated for their ability to stimulate DNA synthesis ([3H]thymidine incorporation) of sparsely seeded quiescent BHK-21 (BHK) cells as well as neonatal rat aortic smooth muscle cells (NRSMC). Conditioned media from monocytes incubated in the presence of beta-VLDL enhanced [3H]thymidine incorporation into DNA of both BHK and NRSMC, when compared to conditioned media harvested from monocytes incubated in the absence of beta-VLDL. Studying NRSMC, this effect was evident using media collected from monocytes incubated with lipoprotein for 2 days; however, a longer incubation of monocytes plus lipoprotein was necessary to induce changes in growth potentiating activity for BHK cells. Likewise, the effect of beta-VLDL treatment of thioglycollate broth elicited BALB/c mouse peritoneal macrophages was evaluated. Conditioned media from lipoprotein-treated macrophages exhibited greater growth-stimulating activity for both BHK cells and NRSMC when compared to conditioned media from macrophages incubated in the absence of the lipoprotein. beta-VLDL did not affect viability of the mononuclear cells. These findings further implicate the involvement of the monocyte-derived foam cell in the development of atherosclerosis.

Limitations on Leaf Nitrate Reductase Activity during Flowering and Podfill in Soybean.

The objective of this study was to identify factors which limit leaf nitrate reductase (NR) activity as decline occurs during flowering and beginning seed development in soybean (Glycine max [L.] Merr. cv Clark). Level of NR enzyme activity, level of reductant, and availability of NO(3) (-) as substrate were evaluated for field-grown soybean from flowering through leaf senescence. Timing of reproductive development was altered within one genotype by (a) exposure of Clark to an artificially short photoperiod to hasten flowering and podfill, and (b) the use of an early flowering isoline. Nitrogen (N) was soil-applied to selected plots at 500 kilograms per hectare as an additional variable. Stem NO(3) (-) concentration and in vivo leaf NR activity were significantly correlated (R(2) = 0.69 with nitrate in the assay medium and 0.74 without nitrate in the medium at P = 0.001) across six combinations of reproductive and soil N-treatment. The supply of NO(3) (-) from the root to the leaf tissue was the primary limitation to leaf NR activity during flowering and podfill. Levels of NR enzyme and reductant were not limiting to leaf NR activity during this period.