Selective cholesteryl ester uptake from high density lipoprotein by human first trimester and term villous trophoblast cells.

As villous trophoblast does represent the contact zone between foetal and maternal tissues, the present in vitro study was aimed at investigating cholesterol supply from human high density lipoprotein subclass 3 (HDL(3)) to trophoblast cells isolated from human first trimester and term placenta. Binding of (125)I-HDL(3) was specific and saturable with similar K(d)-values for first trimester (54 microg HDL(3)-protein/ml) and term villous trophoblast cells (29 microg HDL(3)-protein/ml). The cell-association of (125)I-HDL(3) was 3-fold higher for term trophoblast cells while the specific cell-association of [(3)H]cholesterol ester(CE)-labelled HDL(3) was higher for first trimester trophoblast preparations. As a consequence, first trimester trophoblast cells have a pronounced capacity for selective CE-uptake from HDL(3). Competition experiments with native and oxidized low-density lipoprotein as well as cAMP-mediated stimulation of cell-association of [(3)H]CE-HDL(3) in both trophoblast preparations suggested the scavenger receptor class B, type I (SR-BI) as a likely receptor mediating this pathway. SR-BI m RNA could be identified by RT-PCR and Northern blot experiments in both trophoblast preparations. Western blot analysis and immunocytochemistry revealed high expression of SR-BI in first trimester trophoblast. A polyclonal antiserum raised against murine SR-BI significantly decreased cell-association of [(3)H]CE-HDL(3) in trophoblast cells. We conclude that human first trimester and term trophoblast cells express SR-BI which could serve as an efficient route for supplying cholesterol esters from maternal lipoproteins to foetal tissues.

Gene therapy for cardiovascular disease.

Cardiovascular gene therapy has progressed to the point where clinical trials are now underway, in particular, for peripheral and myocardial ischemia and for restenosis. The delivery vectors are now more sophisticated--targeted to deliver genes only to specific tissues, and with gene expression regulated by disease-inducible promoters--which contributes to improved safety.

The T lymphocyte structure CD60 contains a sialylated carbohydrate epitope that is expressed on both gangliosides and glycoproteins.

The CD60 antigen is expressed on a majority of T cells in autoimmune lesions, and anti-CD60 can activate T lymphocytes. CD60 has been defined as the GD3 ganglioside, and subsequently as the 9-O-acetylated form of GD3. However, other evidence suggests that anti-CD60 recognizes a glycoprotein or family of glycoproteins expressed by T lymphocytes. The current studies were undertaken to better define the identity of the CD60 antigen on both T cells and non-T cells. Treatment of intact cells with neuraminidases of various specificities confirmed that detection of the CD60 epitope depends on expression of an alpha2, 8-disialic acid carbohydrate linkage, as is found in GD3 and related gangliosides. However, the sialicacid polymer colominic acid inhibited anti-GD2 and anti-GD3, but not anti-CD60 from binding to cell surfaces. Expression of CD60 did not correlate with expression of GD3 on a variety of cell lines and T cell populations. Expression of CD60 and 9-O-acetyl-GD3 was roughly parallel on some non-T cell lines such as melanoma cells, but on T cells expression of CD60 was consistently greater. Antibodies to GD2, GD3 and 9-O-acetyl-GD3 were ineffective at inhibiting binding of anti-CD60 to CD60+ cells. Activation responses of T cells to anti-CD60 were inducible in either the presence or absence of a response to anti-GD3. A novel inhibitor of glucosyl ceramide synthesis, D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (D-t-P4) reduced expression of GD3 much more than CD60 on activated T lymphocytes. Following biotinylation of HUT78 T cells, anti-CD60 immunoprecipitated a 70 kDa antigen. Taken together, the present data and previous findings suggest that anti-CD60 can recognize both a modified form of the GD3 ganglioside and a carbohydrate-dependent complex epitope present on one or more glycoproteins. This glycoprotein epitope may be the more abundant and functionally significant CD60 antigen on T lymphocytes, while 9-O-acetyl-GD3 is likely to be the principal structure recognized by anti-CD60 on melanoma cells. These findings emphasize the complexity of understanding the functional roles of carbohydrate epitopes in cell activation.

Transcytosis of lipoprotein lipase across cultured endothelial cells requires both heparan sulfate proteoglycans and the very low density lipoprotein receptor.

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of circulating lipoprotein triglyceride molecules, is synthesized in myocytes and adipocytes but functions while bound to heparan sulfate proteoglycans (HSPGs) on the luminal surface of vascular endothelial cells. This requires transfer of LPL from the abluminal side to the luminal side of endothelial cells. Studies were performed to investigate the mechanisms of LPL transcytosis using cultured monolayers of bovine aortic endothelial cells. We tested whether HSPGs and members of the low density lipoprotein (LDL) receptor superfamily were involved in transfer of LPL from the basolateral to the apical side of cultured endothelial cells. Heparinase/heparinitase treatment of the basolateral cell surface or addition of heparin to the basolateral medium decreased the movement of LPL. This suggested a requirement for HSPGs. To assess the role of receptors, we used either receptor-associated protein, the 39-kDa inhibitor of ligand binding to the LDL receptor-related protein and the very low density lipoprotein (VLDL) receptor, or specific receptor antibodies. Receptor-associated protein reduced (125)I-LPL and LPL activity transfer across the monolayers. When the basolateral surface of the cells was treated with antibodies, only anti-VLDL receptor antibodies inhibited transcytosis. Moreover, overexpression of the VLDL receptor using adenoviral-mediated gene transfer increased LPL transcytosis. Thus, movement of active LPL across endothelial cells involves both HSPGs and VLDL receptor.

Gene delivery to the liver.

Viral gene transfer to the liver has proven extremely effective in animal models and is currently being evaluated in clinical trials for a variety of metabolic disorders. In rodents, a single tail vein injection of an adenoviral vector can transduce most hepatocytes in vivo. This provides a convenient model for assessing vector design as well as for evaluating the effects of specific transgenes in genetic mouse models of human disease. Protocols for optimized in vivo hepatic gene transfer are described.

Sterol carrier protein 2 gene transfer changes lipid metabolism and enterohepatic sterol circulation in mice.

BACKGROUND & AIMS: Sterol carrier protein 2 (SCP-2) enhances sterol cycling and facilitates cholesterol translocation between intracellular organelles and plasma membrane in cultured cells, including hepatocytes. We examined the role of SCP-2 in hepatic cholesterol and lipid trafficking through the sinusoidal and canalicular secretory pathways of the liver in vivo. METHODS: Recombinant adenovirus-mediated SCP-2 gene transfer was used to obtain hepatic overexpression of SCP-2 in C57BL/6 mice. RESULTS: SCP-2 overexpression in the mouse liver resulted in an 8-fold increase of SCP-2 protein levels and determined various effects on lipid metabolism. It decreased high-density lipoprotein cholesterol and increased low-density lipoprotein (LDL) cholesterol concentrations. The expressions of hepatic LDL receptor, apolipoprotein (apo) A-I, apoB, and apoE were decreased. SCP-2 overexpression also increased hepatic cholesterol concentration, associated with decreased cholesterol neosynthesis. Increased biliary cholesterol and bile acid secretion, bile acid pool size, and intestinal cholesterol absorption were also observed. CONCLUSIONS: These results indicate that modulation of SCP-2 expression in the liver determines important modifications on lipoprotein metabolism, hepatic cholesterol synthesis and storage, biliary lipid secretion, bile acid metabolism, and intestinal cholesterol absorption.

Scavenger receptor class B, type I-mediated [3H]cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding to the receptor.

The murine scavenger receptor class B, type I (mSR-BI) is a receptor for high density lipoprotein (HDL), low density lipoprotein (LDL), and acetylated LDL (AcLDL). It mediates selective uptake of lipoprotein lipid and stimulates efflux of [(3)H]cholesterol to lipoproteins. SR-BI-mediated [(3)H]cholesterol efflux was proposed to be independent of ligand binding. In this study, using anti-mSR-BI antibody KKB-1 and two mSR-BI mutants with altered ligand binding properties, we demonstrated that SR-BI-mediated [(3)H]cholesterol efflux to lipoproteins was correlated with ligand binding and lipid uptake activities of the receptor. The KKB-1 antibody, which blocked lipoprotein binding without substantially altering the cholesterol oxidase-accessible cellular [(3)H]cholesterol, also blocked [(3)H]cholesterol efflux to HDL and LDL. One of the SR-BI mutants, which has a double substitution of arginines for glutamines at positions 402 and 418 (Q402R/Q418R), exhibited a high level of LDL binding and lipid uptake from LDL, but lost most of the corresponding HDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to LDL, but not to HDL. Another mutant, M158R, with an arginine in place of methionine at position 158, exhibited reduced HDL and LDL receptor activities, but apparently normal AcLDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to AcLDL, but not to HDL or LDL. These results suggest that SR-BI-stimulated [(3)H]cholesterol efflux to lipoproteins critically depends on ligand binding to this receptor and raise the possibility that the mechanisms of selective lipid uptake and [(3)H]cholesterol efflux may be intimately related.

Gene transfer and hepatic overexpression of the HDL receptor SR-BI reduces atherosclerosis in the cholesterol-fed LDL receptor-deficient mouse.

HDL cholesterol levels in humans are inversely correlated with the risk of atherosclerosis. The class B scavenger receptor type I (SR-BI) is the first molecularly well-defined HDL receptor, and hepatic overexpression of SR-BI in normal mice has been shown to result in decreased plasma HDL cholesterol levels. To determine whether SR-BI overexpression is proatherogenic or is protective against atherosclerosis, LDL receptor-deficient mice were placed on a high-fat/high-cholesterol diet for 2 or 12 weeks to induce atherosclerotic lesions of different stages and then were injected with a recombinant adenovirus encoding murine SR-BI. Transient hepatic overexpression of SR-BI in mice with both early and advanced lesions significantly decreased atherosclerosis. SR-BI expression was associated with markedly decreased HDL cholesterol and either unchanged or only modestly reduced non-HDL cholesterol levels; in all experiments, the mean HDL cholesterol levels were significantly correlated with atherosclerotic lesion size. These data suggest that interventions that promote HDL cholesterol transport and lower plasma HDL cholesterol levels can suppress atherosclerosis, even when initiated after significant lesion development. Thus, stimulation of hepatic SR-BI activity may provide a novel target for therapeutic intervention in atherosclerotic cardiovascular disease.

The HDL receptor SR-BI: a new therapeutic target for atherosclerosis?

Although high-density lipoprotein (HDL) metabolism is a crucial process for cholesterol homeostasis and coronary heart disease, therapeutic approaches for selective modification of plasma HDL levels are not currently available. The discovery of well-defined cell-surface HDL receptors should provide new avenues for treatment of atherosclerotic cardiovascular disease. In fact, SR-BI, a recently identified receptor for selective HDL cholesterol uptake, is relevant for physiological processes (for example, HDL metabolism, steroidogenesis and biliary cholesterol secretion) and pathophysiological conditions (for example, atherosclerosis) in animal models. If SR-BI has similar activities in humans, it might represent a new therapeutic target for atherosclerosis.

Influence of the HDL receptor SR-BI on atherosclerosis.

The scavenger receptor class B, type I (SR-BI) is an HDL receptor that mediates selective cholesterol uptake from HDL to cells. In rodents, SR-BI has a critical influence on plasma HDL-cholesterol concentration and structure, the delivery of cholesterol to steroidogenic tissues, female fertility, and biliary cholesterol concentration. SR-BI can also serve as a receptor for non-HDL lipoproteins and appears to play an important role in reverse cholesterol transport. Recent studies involving the manipulation of SR-BI expression in mice, either using adenovirus-mediated or transgenic hepatic overexpression or using homologous recombination for complete functional ablation, indicate that the expression of SR-BI protects against atherosclerosis. If SR-BI has a similar activity in humans, it may become an attractive target for therapeutic intervention.

Localization of the steroidogenic acute regulatory protein in human tissues.

The rate-limiting step in steroid hormone production in the adrenal cortex and gonads, the translocation of cholesterol from the outer to the inner mitochondrial membranes, is mediated by the steroidogenic acute regulatory protein (StAR). Heretofore, the localization of StAR in human adult and fetal tissues has not been defined. To this end, expression of StAR was detected in formalin-fixed, paraffin-embedded specimens using a polyclonal antiserum raised against recombinant human StAR. Primordial follicles of adult ovaries did not contain StAR, whereas antral follicles stained intensely in the thecal layer, with occasional staining of granulosa cells. Corpora lutea were intensely stained, but with a patchy distribution. Corpora albicantia did not stain. A luteoma of pregnancy stained with patches of moderate intensity. Ovaries with hyperthecosis contained areas of intense thecal staining. An ovarian Leydig cell tumor stained intensely, whereas granulosa cell tumors were negative. Ovarian adenocarcinomas, borderline tumors, teratomas, cystadenomas, and a Brenner tumor displayed no specific StAR immunostaining. Testicular Leydig cells stained moderately to intensely, as did a testicular Leydig cell tumor. Sertoli cells stained weakly in some specimens. Seminomas and testicular germ cell tumors were negative. There was minimal to moderate staining in the adrenal glomerulosa and faciculata and minimal staining in the reticularis, while the medulla was negative. Adrenal cortical adenomas, hyperplasias, and carcinomas all contained areas of StAR staining. The renal distal tubules stained with moderate to marked intensity. Renal carcinomas had occasional modest staining. No immunostaining was found in the placenta. Fetal ovaries contained sporadic stromal cells displaying intense StAR staining, particularly in the hilar region. Oocytes from a 32-week fetal ovary showed moderate to intense staining. Fetal testes displayed intense Leydig cell staining. The neocortex of the fetal adrenal glands displayed only minimal StAR staining, whereas moderate to intense staining was found in the fetal zone. The fetal kidneys had moderate StAR staining of the distal convoluted tubules. We conclude that StAR is localized to normal and neoplastic cells in the gonads and adrenal cortex, which produce large amounts of pregnenolone. StAR protein was not detected in the placenta, documenting that placental progestin synthesis occurs through StAR-independent mechanisms. The presence of StAR in cells that do not express cholesterol side-chain cleavage enzyme cytochrome P450, including renal distal tubules, Sertoli cells, and fetal oocytes, suggests that StAR has roles in metabolic processes in addition to stimulating pregnenolone synthesis.

The atherogenic lipoprotein Lp(a) is internalized and degraded in a process mediated by the VLDL receptor.

Lp(a) is a major inherited risk factor associated with premature heart disease and stroke. The mechanism of Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to influence plasminogen activation as well as its atherogenic potential as a lipoprotein particle after receptor-mediated uptake. We demonstrate that fibroblasts expressing the human VLDL receptor can mediate endocytosis of Lp(a), leading to its degradation within lysosomes. In contrast, fibroblasts deficient in this receptor are not effective in catabolizing Lp(a). Lp(a) degradation was prevented by antibodies against the VLDL receptor, and by RAP, an antagonist of ligand binding to the VLDL receptor. Catabolism of Lp(a) was inhibited by apolipoprotein(a), but not by LDL or by monoclonal antibodies against apoB100 that block LDL binding to the LDL receptor, indicating that apolipoprotein(a) mediates Lp(a) binding to this receptor. Removal of Lp(a) antigen from the mouse circulation was delayed in mice deficient in the VLDL receptor when compared with control mice, indicating that the VLDL receptor may play an important role in Lp(a) catabolism in vivo. We also demonstrate the expression of the VLDL receptor in macrophages present in human atherosclerotic lesions. The ability of the VLDL receptor to mediate endocytosis of Lp(a) could lead to cellular accumulation of lipid within macrophages, and may represent a molecular basis for the atherogenic effects of Lp(a).

Overexpression of the HDL receptor SR-BI alters plasma HDL and bile cholesterol levels.

The risk of atherosclerosis, a leading cause of cardiovascular disease and death, is inversely related to plasma levels of high-density lipoprotein (HDL) cholesterol, although the mechanism of this protective effect is unclear. The class B scavenger receptor, SR-BI, is the first HDL receptor to be well defined at a molecular level and is a mediator of selective cholesterol uptake in vitro. It is expressed most abundantly in steroidogenic tissues, where it is coordinately regulated with steroidogenesis by adrenocorticotropic hormone (ACTH), human chorionic gonadotropin (hCG) and oestrogen, and in the liver, where its expression in rats is suppressed by oestrogen. Here we show that adenovirus-mediated, hepatic overexpression of SR-BI in mice on both sinusoidal and canalicular surfaces of hepatocytes results in the virtual disappearance of plasma HDL and a substantial increase in biliary cholesterol. SR-BI may directly mediate these effects by increasing hepatic HDL cholesterol uptake or by increasing cholesterol secretion into bile, or both. These results indicate that SR-BI may be important in hepatic HDL metabolism, in determining plasma HDL concentrations, and in controlling cholesterol concentrations in bile, and thus may influence the development and progression of atherosclerosis and gallstone disease.

Effective treatment of familial hypercholesterolaemia in the mouse model using adenovirus-mediated transfer of the VLDL receptor gene.

Liver directed gene transfer with adenoviral vectors is being considered for the treatment of several metabolic diseases, including familial hypercholesterolaemia (FH). Gene replacement therapy of human low density lipoprotein (LDL) receptor gene into the murine model of FH transiently corrected the dyslipidaemia; however, humoral and cellular immune responses to LDL receptor developed--possibly contributing to the associated hepatitis and extinguishing of transgene expression. We evaluated an alternative strategy of ectopic expression in the liver of the very low density lipoprotein (VLDL) receptor, which is homologous to the LDL receptor but has a different pattern of expression. Infusion of recombinant adenoviruses containing the VLDL receptor gene corrected the dsylipidaemia in the FH mouse and circumvented immune responses to the transgene leading to a more prolonged metabolic correction.

Hepatic expression of the catalytic subunit of the apolipoprotein B mRNA editing enzyme (apobec-1) ameliorates hypercholesterolemia in LDL receptor-deficient rabbits.

Apolipoprotein (apo) B48, a protein contained in intestinally derived lipoprotein particles, is synthesized by post-transcriptional editing of apoB100 mRNA. This reaction is mediated by an enzyme complex that includes the catalytic subunit, apobec-1. The liver of most mammals, by contrast, contains only unedited apoB mRNA and secretes apoB100, the major protein component of plasma low-density lipoprotein (LDL). Because rabbits, like humans, fail to edit hepatic apoB100 mRNA, we introduced a recombinant adenovirus encoding apobec-1 into the livers of LDL receptor-defective rabbits to determine the impact on lipoprotein metabolism of hepatic apoB48 secretion. Transgene expression was mainly confined to the liver and was sustained for up to 3 weeks following virus administration, as evidenced by the presence of apobec-1 mRNA and the ability of hepatic S100 extracts to edit a synthetic apoB RNA template in vitro. The transient induction of hepatic apoB mRNA editing accompanied alterations in very-low-density lipoprotein (VLDL) size, the presence of apoB48 in fractions spanning the VLDL and LDL range, and modest reductions in total plasma cholesterol levels.

Transduction of human trophoblast cells by recombinant adenoviruses is differentiation dependent.

To explore the feasibility of adenoviral (Ad)-mediated gene transfer to the human placenta, we examined the ability of two recombinant Ad vectors to transduce isolated human cytotrophoblast cells and two choriocarcinoma cell lines (BeWo and JEG-3, which have different potentials to undergo morphological differentiation in response to cAMP). Recombinant Ad efficiently transduced cytotrophoblast cells. However, there was a marked reduction in the transduction efficiency of these vectors after the terminal differentiation of the mononucleate cytotrophoblasts into multinucleate syncytial trophoblast. BeWo and JEG-3 cells were readily transduced with the recombinant Ad, but a striking reduction in transduction efficiency of the Ad vector was observed in BeWo cells following cAMP-stimulated cellular differentiation, which includes cell fusion to form syncytia. In contrast, JEG-3 cells, which are not induced to fuse in the presence of cAMP, did not show a reduced transduction efficiency when exposed to the cyclic nucleotide. Reporter gene copy number increased with Ad-mediated gene transfer into undifferentiated Bewo cells but was low in cells that had been previously exposed to cAMP. In contrast, both undifferentiated and cAMP-treated BeWo cells were capable of expressing a reporter gene when transfected with an Ad-based plasmid. Taken together, these results demonstrate that the reduction in transduction efficiency of the Ad vectors in cAMP-treated BeWo is the result of reduced infectivity rather than of a reduction in the transcription/translation efficiency of the exogenous genes. Our findings demonstrate that recombinant Ad vectors will not be useful for the transfer of genes into differentiated trophoblast cells because these cells are resistant to Ad infection. This may limit the utility of Ad-based vectors for placental gene therapy. However, we have documented that less-differentiated trophoblast cells are susceptible to Ad-mediated gene transfer. Our observations also suggest a mechanism by which differentiated human trophoblast cells resist Ad infection and prevent fetal infection by maternally derived Ad.

Prolongation of adenoviral transgene expression in mouse liver by T lymphocyte subset depletion.

Although first generation recombinant adenoviruses are efficient vehicles for gene transfer, their immunogenicity precludes long-term persistence. We show that adenoviral transgene expression in the liver of normal mice is prolonged from a baseline of less than 2 weeks to 7 weeks by depleting CD4+ T lymphocytes with thymectomy and a 3-day course of anti-CD4 monoclonal antibody or by nonselectively depleting T cells with a single dose of anti-thymocyte serum (ATS). Transgene expression persisted despite the development of an antiviral humoral immune response by 3 weeks after virus administration. In vitro assays of T lymphocyte function revealed an initial diminished capacity to proliferate in the presence of adenoviral antigens in animals depleted of CD4+ T cells or given anti-thymocyte serum. Eventual loss of recombinant gene expression coincided with the development of adenovirus-specific cytotoxic T lymphocyte activity in vitro. Immunosuppression provides a useful experimental tool to elucidate the immunobiology of recombinant adenoviruses and may have clinical application to adenovirus-mediated gene therapy.

A method for monoclonal antibody isotype switching: anti-CD60 VH expression in a heavy chain-deficient hybridoma variant.

Alteration of monoclonal antibody isotype is desirable for a variety of purposes, including obtaining an improved reagent for investigative or therapeutic use. A variety of approaches for isotype switching, particularly from IgM to various IgG subclasses, have been described. Antibodies that recognize carbohydrate determinants on glycoproteins, glycolipids, or polysaccharides are generally of the IgM isotype. This includes all available antibodies to the human CD60 antigen, a determinant with cell coactivating properties described on a subset of T lymphocytes and on other cell types. In this report a new method for monoclonal antibody isotype switching is presented. A plasmid containing the VH regions of anti-CD60 linked to C gamma 1 was transfected into a spontaneously arising variant of the CD60 hybridoma that produced kappa light chain but no heavy chain. This transfected hybridoma line maintains stable production of useful quantities of IgG1 monoclonal anti-CD60 in vitro and in vivo.

A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolaemia.

The outcome of the first pilot study of liver-directed gene therapy is reported here. Five patients with homozygous familial hypercholesterolaemia (FH) ranging in age from 7 to 41 years were enrolled; each patient tolerated the procedure well without significant complications. Transgene expression was detected in a limited number of hepatocytes of liver tissue harvested four months after gene transfer from all five patients. Significant and prolonged reductions in low density lipoprotein (LDL) cholesterol were demonstrated in three of five patients; in vivo LDL catabolism was increased 53% following gene therapy in a receptor negative patient, who realized a reduction in serum LDL equal to approximately 150 mg dl-1. This study demonstrates the feasibility of engrafting limited numbers of retrovirus-transduced hepatocytes without morbidity and achieving persistent gene expression lasting at least four months after gene therapy. The variable metabolic responses observed following low-level genetic reconstitution in the five patients studied precludes a broader application of liver-directed gene therapy without modifications that consistently effect substantially greater gene transfer.