Differential expression and stability of scavenger receptor isoforms in human monocyte-derived macrophages

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Wednesday 12 October 1994: Poster Abstracts Cell biology

receptor, continued to metabolize remnants. In vivo chylomicron remnants am primarily cleared via the LDL [l] receptor so we wanted to determine the mechanism of remnant uptake by macrophages. Competition studies with unlabeled chylomicron remnants showed that only about one-third of total degradation was via high affinity (receptor) mechanisms. Unlabeled LDL also inhibited remnant binding and degradation by about 301, suggesting involvement of the LDL receptor. However, degradation in the absence of Ca2+ and presence of EDTA, or in the presence of the lysosomal inhibitors colchicine and chloroquine, had no significant effect on the binding or degradation of remnants by macrophages. Competition with inhibitors of scavenger receptors, namely fucoidan or polyinosinic acid, decreased uptake by only a modest 30%. Consistent with a nominal role of high affinity mechanisms in remnant metabolism by macrophages, cytochalasin D, which blocks phagocytosis, had a substantial effect on chylomicron remnant binding and degradation (65% inhibition). Collectively, our data show that chylomicron remnants can bind to and be internalized by both the LDL receptor and scavenger receptors; however, the primary mute of chylomicron remnant uptake is via phagocytosis. Uptake of remnants was typically greater than that by LDL, which suggests that the phagocytotic mechanism was selective. Chylomicron remnants inhibited cholesterol biosynthesis and increased cholesterol esterification in alveolar macrophages. The effects were quantitatively similar to LDL but substantially less than beta VLDL. Limited data suggested that intracellular events may differ for macrophages isolated from Watanabe rabbits. Chylomicron remnants serve as an excellent substrate for macrophages and display metabolic characteristics consistent with their putative role in atherogenesis. [l] Bowler A, Redgrave TG, Mamo JCL Biochem J 1991; 276: 381-386.

It has been demonstrated that complement components and recep tars exist in atherosclerotic lesions and the complement system might be related to the initiation and promotion of atherosclerosis. The aim of the present investigation was to examine the influence of LDL or Cu2+-oxidized LDL on the secretion of C3 by human monocyte-macrophages. An immunohistological study of consecutive aortic sections was performed for macrophages and iC3b, employing the alkaline phosphatase/anti-alkaline phosphatase method. Human mononuclear cells were isolated from peripheral blood by Ficoll-Paque gradient, and monocytes were then separated from non-adherent cells to plastic dishes. We used the monocytes cultured for 7 days with RPM11640 containing 10% fetal calf serum as macrophages. After the culture, they were coincubated with LDL, oxidized LDL or lipopolysaccharide (LPS) in the serum-free medium for 24 h. C3 biosynthesis by the macrophages was demonstrated by direct immunostaining and the incorporation of [35S]methionine into immunoprecipitable complement proteins. We further investigated the production of C3 by the macrophages employing competitive ELISA. In consecutive aortic sections with intimal thickening, iC3b deposits were noted around the macrophage antigen-positive cells. In vitro studies indicated that oxidized LDL stimulated C3 biosynthesis and protein production by cultured human macrophages far more than did LDL or LPS. In conclusion, the C3 present in atherosclerotic lesions may be partially produced by macrophages, being stimulated by oxidized LDL or LDL. iC3b, i.e. a decay product of C3 and the ligand of complement receptor type 3(CR3), occurs around the macrophages in the atherosclerotic lesions. Lymphocyte T subset counts in children at risk for 1 atherosclerosis m, Morcno L, Mur M, L&aro A, Lasierca MP, Roda L, Giner A, Larrad L, Bueno M, Dept. de Pediatn’a, Hospital Clinic0 Univ., Avda. San Juan Bosco 15, 50009 Zaragoza, Spain

Influence of modified LDL on gap junctional com1 munication between human aortic intimal cells ,4ndreeva ER, Serebryakov VN, Orekhov AN, Inst. of Experimental Cardiol., National Cardiol. Res. Center, 3rd Cherepkovskaya str 15A, 121552 Moscow, Russia

To investigate the role of gap junctional communication in human atherosclerosis we used the model of modified LDL-induced lipid accumulation in culture of human aortic intimal cells. Primary cultures of human aortic cells from normal regions were obtained by standard procedures. On the 7th day of culture native LDL, desialylated or oxidized LDL (100pglml) was added. After 18 h of incubation intercellular communication was assessed by evaluating the transfer of the microinjection fluorescent dye calcein between cultured cells (standard glass microelectrade, resistance 70-100 MQ, and pulse injection for 5 s). The number of fluorescent labeled cells per injection (c/i) increased linearly with cell density up to 60 cells/mm2 and then remained constant at about 20.3 & 1.3 c/i. Cells with native LDL achieved 18.9 f 1.l c/i, significantly more than the cell-tocell coupling seen between cells incubated with oxidized LDL (9.9 f 0.9) or desialylated LDL (8.5 + 0.6 c/i). We suggest that the decreased permeability of gap junctions induced by modified LDL may be explained by increased lipid accumulation in aortic cells.

The objective of this study was to determine peripheral lymphocyte T subset counts in children with high serum LDL-C. We studied 107 children, 2.0-15.9 years, from 79 families referred to our Lipid Research Clinic because TC > 200 mg/dl had been detected in at least one child. We analyzed serum lipoprotein profile and blood lymphocyte T subsets (CD3, CD4 and CD8). Children were classified by LDL-C level into three groups: normal, if levels were between the 5th and 75th percentiles (50 and 125 mg/dl), at moderate risk, levels between 75th and 95th percentiles (125 and 150 mg/dl), and at high risk, if levels were above 150 mg/dl. In children 2.0-6.9years. all lymphocyte T subset counts were higher in the high-risk group than in the normal group (P < 0.05 to P < 0.01); CD3 and CD4 subset counts showed significant correlations with TC, LDL-C, apo A-I and apo B (P < 0.05 to P < 0.01). In children 11.0-15.9 years, the CD4 subset count was also significantly higher in the high-risk group than in the other two groups (P < 0.05 and P < 0.01); CD3, CD4 and CD8 showed significant correlations with serum triglyceride levels (P < 0.05 and P < 0.01). These results are in agreement with pathological findings in the atheromatous plaque. 1681 w,

Differential expression and stability of scavenger receptor isoforms in human monocyte-derived macrophages Giroux LM, Roy M, Minnich A, Davignon J, Atheroscle-

El

rosis and Hyperlipidemia, Clin. Res. Inst. of Montreal, 110 Pine Ave West, Montreal, PQ, Canada H2W lR7

Med., Nihon Univ. Sch. of Med., 30-I Ohyaguchi Kami-machi, Itabashi-ku, Tokyo I73, Japan

Macrophage scavenger receptor (SR) is widely suspected to play a role in foam cell formation and subsequently in atherogenesis by promoting lipoprotein influx and cellular sequestration of cholesterol into resident macrophages in a non-sterol regulated manner.

Intluence of oxidized LDL on secretion of the third component of complement (C3) by cultured human peripheral monocyte-derived macrophages &&& Fujioka T, Kanno H, Ueno T, Matsumoto T, Takahashi Y, Watanabe H, Tochihara T, Kanmatsuse K, 2nd Dept. of Int.

Atherosclerosis X, Montreal, October 1994

Wednesday 12 October 1994: Poster Abstracts Geneiics

We have previously reported (Giry et al Circulation 1993; 88: 133) a case of familial SR overexpression associated with planar xanthomas in the absence of hyperlipidemia. This unusual abnormality prompted us to look at mechanisms regulating SR expression. Based on fluorescent RT-PCR quantification of mRNA, our data showed that SR isoform 1 was predominantly expressed during monocytic maturation in patients as well as in controls. Relative contribution of SRI to the whole SR message was 60% in freshly isolated monocytes and 80% in monocyte-derived macrophages. However, SR message including both isoforms was 510 times higher in l-day monocytes from patients than in controls. Patients’ monocytes expressed 42% more SR mRNA than controls after 4 days of maturation, and 65% after 7 days. In foam

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cells obtained after 12 days of culture, SR expression was 14.8 times that of controls. Stability of SR message was also determined in monocytes after 4 days in culti using actinomycin D. In control monocytes, SR messages (forms 1 and 2 together) were more stable (tin= 235min) than SR2 mRNA (tta=68min), while in two affected siblings the messages of SR (forms 1 and 2) and SR2 were highly unstable (tla = 62 and 41 min respectively). These results indicate a posttranscriptional regulation of SR based on mRNA stability, which may be specific for SR isoform 1 and may be associated with high levels of SR gene transcription. This phenomenon needs to be confirmed in situations where the scavenger receptor is up-regulated.

GENETICS

1

Analysis of the 5’ region of apo A-I gene in subjects with low HDL-C Civeira F, Gonzaez J, Ordovb JM, Pocovi M, Dept. mA, Bioqui’mica, Biologia-Molecular, Univ. Zaragoza, 50009 Saragoza, Spain

Hepatic synthesis of apo A-I is one of the mechanism that regulates apo A-I plasma levels, and is directly related to hepatic apo A-I gene transcription. One 215 bp fragment located in the 5’ region of the gene is sufficient for maximal expression in hepatic cell lines. It has been suggested that mutations in this sequence can modify transcription and apo A-I plasma levels. The objective of this study was to determine possible mutations in the promoter region of apo A-I, in a group of subjects with primary low HDLC levels. 41 males with HDL-C c 35 mg/ dl in at least four different analyses were included. Exclusion criteria were: TC or TG >90th percentile, diabetes mellitus, obesity (BMI > 30) or cigarette smoking. A DNA fragment (259 bp) in the promoter region of apo A-I which included the mentioned 215 fragment was amplified with Taq DNA polymerase. Mutation detection was done by thermal gradient electrophoresis and by heteroduplex detection method in MDE gels (AT B&hem). Both methods are based on migration differences between wild and mutant allele, and detected the same results: 14 (34%) subjects heterozygous for this DNA fragment. All of them were heterozygous for a G/A substitution in position -78. This polymorphism has been previously described and is not clearly related to low HDL-C concentrations. In conclusion, the -78 G/A substitution is the only mutation that was found in this 259 fragment in apo A-I gene promoter region in subjects with primary low HDL-C. This DNA fragment does not seem to be related to the mechanism of low HDL-C concentrations, at least in this study. 1

Identification of a novel mutation (Argw + Cys) in the LCAT gene causing Fish Eye Disease in a Spanish

family t 2 , Qu S-J1, Fiol C31 Fan H-Z1, Marzal. Hurtado 13. Gracia V . Pint6 X3. Marti T3, Albers JJ4, Pow&l HJt, ’B&or Coil. ifMed., H&on, TX:

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USA; ‘Inst. de Recerca de I’Hospiral de la Santa Creu i Sant Pau (Bioquimica) i Universitaf Autcinoma de Barcelona. C/ Antoni M. Claret 167, 08025 Barcelona, Spain; 3Ciutat Sanitciria i Universitciria de Bellvitge, L’Hospitalet de Llobregat, Spain: ‘Univ. of Washington Sch. of Med., Seattle, WA, USA

Inherited HDL deficiencies are good models in which to investigate the structure and functions of HDL and their relationship to atherosclerosis. The molecular bases of inherited HDL deficien-

cies are therefore of interest. We had the opportunity to study a 68-year-old woman with the clinical and biochemical characteristics of a very infrequent HDL deficiency called Fish Eye Disease (FED). The patient presented with comeal opacities, very low plasma HDL-C, a decreased concentration of cholesteryl esters in HDL whereas that in apo B-containing lipoproteins was normal, and low plasma LCAT activity. The DNA sequence of the exons of the LCAT gene amplified by PCR showed hitherto an undescribed mutation in amino acid 99. This change was not present in control human genomic DNA. The mutation generated a new restriction site for BglII. RFLP analysis using BgiII showed that the patient was homozygous for the mutation and that her three daughters, all of whom had low plasma HDL-C, were heterozygous. Mutagenesis in vitro confirmed that the (Arggg + Cys) mutation was the cause of this case of FED. Apo A-UC-IIYA-IV gene cluster in familial combined 1 hyperlipidemia Dallinea_Thie, Van Linde-Sibenius Trip M, Ploos van Amstel HK, De Bruin TWA, Depts. of Med. and Clin. Generics, Acad. Hosp. Urrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands

Familial combined hyperlipidemia (FCH) is a frequent genetic disorder with increased risk of premature atherosclerosis. Its characteristic multiple phenotypic expression suggests involvement of several genes in the expression of the hyperlipidemia. Recently, presence of the X2 allele was associated with hypertriglyceridemia and insulin resistance (Castro Cabezas et al J Clin Invest 1993; 92: 160-168). The present study focused on the role of the apo A-I/C-III/A-IV gene cluster, located on chromosome 11. Three polymorphic markers were tested: Xmn I, located 2.5 kb upstream of the apo A-I gene; Msp I, located in the promoter region (-78), and Sst I, located in exon 4 of the apo C-III gene. The frequency of the M2, S2, and X2 alleles was significantly higher in FCH subjects (n = 198) than in normolipidemic spouses (n = 121): M2 42.9% vs 27% (P = O.Ol), S2 20.2% vs 9.8% (P = 0.06) and X2 36.9% vs 24.6% (P = 0.06). The plasma lipids were much higher in the FCH group than in their spouses: Chol 7.47 + 2.48 mmol/l vs 5.17 kO.15 mmoV1, and TG 3.69 2 7.29 vs 1.28 + 0.41 mmovl (P < 0.001 each). Interestingly, normolipidemic relatives (Chol4.91 f 0.80 mmol/l and TG 1.22 + 0.38 mmolfl, n = 193) also showed a high marker frequency: M2 36.8%, S2 11.9%, X2 34.2%, but this was not statistically different from the spouses. Potential development of the FCH phenotype in this group cannot be excluded because of the proportion of relatives with age <25 years (n = 74). In conclusion, the apo A-I/C-III/A-IV gene cluster is important for the expression of hyperlipidemia in FCH. It is likely that other gene(s) are also involved.

Atherosclerosis X, Montreal, October 1994