Hypertriglyceridemia as a result of human apo CIII gene expression in transgenic mice.

Primary and secondary hypertriglyceridemia is common in the general population, but the biochemical basis for this disease is largely unknown. With the use of transgenic technology, two lines of mice were created that express the human apolipoprotein CIII gene. One of these mouse lines with 100 copies of the gene was found to express large amounts of the protein and to be severely hypertriglyceridemic. The other mouse line with one to two copies of the gene expressed low amounts of the protein, but nevertheless manifested mild hypertriglyceridemia. Thus, overexpression of apolipoprotein CIII can be a primary cause of hypertriglyceridemia in vivo and may provide one possible etiology for this common disorder in humans.

Plasma Ip(a) concentration is inversely correlated with the ratio of Kringle IV/Kringle V encoding domains in the apo(a) gene.

Plasma Lp(a) levels correlate with atherosclerosis susceptibility. This lipoprotein consists of an LDL-like particle attached to a large glycoprotein called apo(a). Apo(a) is a complex glycoprotein containing multiple Kringle domains, found to be highly homologous to plasminogen Kringle IV, and a single Kringle domain homologous to plasminogen Kringle V. Lp(a) levels appear to be inversely correlated with apo(a) size in a given individual. In this study, we have used probes specific to the Kringles IV and V domains of apo(a) cDNA in quantitative Southern blotting analysis. By this method, we have determined the ratio of Kringle IV/Kringle V encoding domains in the apo(a) gene of 53 unrelated individuals with different plasma concentrations of Lp(a). This ratio was found to be inversely correlated with log Lp(a) levels (r = -0.90, P less than 0.0001) and directly correlated with apo(a) apparent molecular weight (Mr) (r = 0.79, P less than 0.0001). In summary, by showing that Lp(a) concentrations and apo(a) apparent size are highly correlated with the ratio of Kringle IV/Kringle V encoding domains in the apo(a) gene, we provide a DNA marker for this atherosclerosis risk factor as well as an important insight into the genetic mechanism regulating Lp(a) levels.

Dietary fat increases high density lipoprotein (HDL) levels both by increasing the transport rates and decreasing the fractional catabolic rates of HDL cholesterol ester and apolipoprotein (Apo) A-I. Presentation of a new animal model and mechanistic studies in human Apo A-I transgenic and control mice.

In humans, diets high in saturated fat and cholesterol raise HDL-cholesterol (HDL-C) levels. To explore the mechanism, we have devised a mouse model that mimics the human situation. In this model, HuAITg and control mice were studied on low fat (9% cal)-low cholesterol (57 mg/1,000 kcal) (chow) and high fat (41% cal)-high cholesterol (437 mg/1,000 kcal) (milk-fat based) diets. The mice responded to increased dietary fat by increasing both HDL-C and apo A-I levels, with a greater increase in HDL-C levels. This was compatible with an increase in HDL size observed by nondenaturing gradient gel electrophoresis. Turnover studies with doubly labeled HDL showed that dietary fat both increase the transport rate (TR) and decreased the fractional catabolic rate of HDL cholesterol ester (CE) and apo A-I, with the largest effect on HDL CE TR. The latter suggested that dietary fat increases reverse cholesterol transport through the HDL pathway, perhaps as an adaptation to the metabolic load of a high fat diet. The increase in apo A-I TR by dietary fat was confirmed by experiments showing increased apo A-I secretion from primary hepatocytes isolated from animals on the high fat diet. The increased apo A-I production was not associated with any increase in hepatic or intestinal apo A-I mRNA, suggesting that the mechanism of the dietary fat effect was posttranscriptional, involving either increased translatability of the apo A-I mRNA or less intracellular apo A-I degradation. The dietary fat-induced decrease in HDL CE and apo A-I fractional catabolic rate may have been caused by the increase in HDL particle size, as was suggested by our previous studies in humans. In summary, a mouse model has been developed and experiments performed to better understand the paradoxical HDL-raising effect of a high fat diet.

Hypertriglyceridemia and cholesteryl ester transfer protein interact to dramatically alter high density lipoprotein levels, particle sizes, and metabolism. Studies in transgenic mice.

Several types of transgenic mice were used to study the influence of hypertriglyceridemia and cholesteryl ester transfer protein (CETP) expression on high density lipoprotein (HDL) levels, particle sizes, and metabolism. The presence of the CETP transgene in hypertriglyceridemic human apo CIII transgenic mice lowered HDL-cholesterol (HDL-C) 48% and apolipoprotein (apo) A-I 40%, decreased HDL size (particle diameter from 9.8 to 8.8 nm), increased HDL cholesterol ester (CE) fractional catabolic rate (FCR) 65% with a small decrease in HDL CE transport rate (TR) and increased apo A-I FCR 15% and decreased apo A-I TR 29%. The presence of the CETP transgene in hypertriglyceridemic mice with human-like HDL, human apo A-I apo CIII transgenic mice, lowered HDL-C 61% and apo A-I 45%, caused a dramatic diminution of HDL particle size (particle diameters from 10.3 and 9.1 to 7.6 nm), increased HDL CE FCR by 107% without affecting HDL CE TR, and increased apo A-I FCR 35% and decreased apo A-I TR 48%. Moreover, unexpectedly, hypertriglyceridemia alone in the absence of CETP was also found to cause lower HDL-C and apo A-I levels primarily by decreasing TRs. Decreased apo A-I TR was confirmed by an in vivo labeling study and found to be associated with a decrease in intestinal but not hepatic apo A-I mRNA levels. In summary, the introduction of the human apo A-I, apo CIII, and CETP genes into transgenic mice produced a high-triglyceride, low-HDL-C lipoprotein phenotype. Human apo A-I gene overexpression caused a diminution of mouse apo A-I and a change from monodisperse to polydisperse HDL. Human apo CIII gene overexpression caused hypertriglyceridemia with a significant decrease in HDL-C and apo A-I levels primarily due to decreased HDL CE and apo A-I TR but without a profound change in HDL size. In the hypertriglyceridemic mice, human CETP gene expression further reduced HDL-C and apo A-I levels, primarily by increasing HDL CE and apo A-I FCR, while dramatically reducing HDL size. This study provides insights into the genes that may cause the high-triglyceride, low-HDL-C phenotype in humans and the metabolic mechanisms involved.

The full induction of human apoprotein A-I gene expression by the experimental nephrotic syndrome in transgenic mice depends on cis-acting elements in the proximal 256 base-pair promoter region and the trans-acting factor early growth response factor 1.

To identify molecular factors regulating apo A-I production in vivo, we induced in transgenic mice the experimental nephrotic syndrome, which results in elevated levels of HDL cholesterol (HDL-C), plasma apo A-I, and hepatic apo A-I mRNA. Human (h) apo A-I transgenic mice with different length 5' flanking sequences (5.5 or 0.256 kb, the core promoter for hepatic-specific basal expression) were injected with nephrotoxic (NTS) or control serum. With nephrosis, there were comparable (greater than twofold) increases in both lines of HDL-C, h-apo A-I, and hepatic h-apo A-I mRNA, suggesting that cis-acting elements regulating induced apo A-I gene expression were within its core promoter. Hepatic nuclear extracts from control and nephrotic mice footprinted the core promoter similarly, implying that the same elements regulated basal and induced expression. Hepatic mRNA levels for hepatocyte nuclear factor (HNF) 4 and early growth response factor (EGR) 1, trans-acting factors that bind to the core promoter, were measured: HNF4 mRNA was not affected, but that of EGR-1 was elevated approximately fivefold in the nephrotic group. EGR-1 knockout (EGR1-KO) mice or mice expressing EGR-1 were injected with either NTS or control serum. Levels of HDL-C, apo A-I, and hepatic apo A-I mRNA were lowest in nonnephrotic EGR1-KO mice and highest in nephrotic mice expressing EGR-1. Although in EGR1-KO mice HDL-C, apo A-I, and apo A-I mRNA levels also increased after NTS injection, they were approximately half of those in the nephrotic EGR-1-expressing mice. We conclude that in this model, basal and induced apo A-I gene expression in vivo are regulated by the trans-acting factor EGR-1 and require the same cis-acting elements in the core promoter.

Cellular distribution of cholesterogenesis-linked, phosphoisoprenylated proteins in proliferating cells.

A set of isoprenylated proteins has been detected in rapidly proliferating, suspension-grown murine lymphoma cells. Our evidence indicates that all of these isoprenylated proteins are phosphorylated. Subsequent to a 24 h incubation with mevinolin to deplete the intracellular mevalonate (MVA) level, cells were incubated with [3H]MVA and/or 32Pi and both total cell and subcellular fraction proteins were resolved via 1- and 2-D gel electrophoresis, then assessed via subsequent autoradiography. The phospho-isoprenylated proteins comprise a set spanning a molecular mass range of 21-69 kDa and all dispay acidic pI. MVA-derivatized proteins of 21-24 kDa, which consist of multiple isoforms, are present in both cytosolic and nuclear fractions. Larger phospho-isoprenylated protein species (44-69 kDa) are specifically localized within the nucleus, where applicable extraction protocols indicate that they are part of or closely affiliated with the nuclear matrix-intermediate filament (NM-IF) components. The localization of the 69 kDa prenylated species within the NM-IF fraction, together with evidence of its phosphorylation, supports recent indications that this protein is the nuclear matrix component lamin B.

Idea management: restructuring technology-based research to help achieve corporate goals.

In order for some technology-based companies to continue to compete, they must consider changes and improvements in the mechanism(s) by which new ideas are generated and transformed into putative products. The manner in which technology-based research is currently organized in most companies and the multiple demands of the duties assigned to the Group Leader often dictate that new product development initiation is the task to which he or she gives the least attention. A re-design of the technology-based business process of collecting ideas for novel and profitable products is implicit in SCIMAX (short for SCientific Idea Management and Assessment Extension). SCIMAX provides a mechanism for either acting on new ideas or storing them for future reference. Once a putative target is found to be commercially and scientifically valid (according to SCIMAX), as well as technically feasible (by the Exploratory Group), it can be presented to upper management for consideration as a legitimate target. Restructuring technology research to implement an idea management system such as SCIMAX would require a relatively minor effort on part of the corporate management.

A solution hybridization/RNase protection assay with riboprobes to determine absolute levels of apoB, A-I, and E mRNA in human hepatoma cell lines.

A solution hybridization/RNase protection assay with riboprobes was developed to quantitate apolipoprotein mRNA concentrations. Previously, radiolabeled DNA probes have been used in solution hybridization/S1 nuclease protection assays for this purpose. The new assay requires less time for probe preparation and hybridization compared to previous assays. In addition, the vector used for riboprobe preparation can also be used to conveniently produce cRNA required to generate the standard curve to quantitate absolute apolipoprotein mRNA levels. The solution hybridization RNase protection assay was used to quantitate apoB, A-I, and E mRNA levels in four human hepatoma cell lines, HepG2, Hep3B, WRL-68, SK-Hep2. HepG2 and Hep3B, but not WRL-68 and SK-Hep2 cells had concentrations of all three apolipoprotein mRNAs comparable to liver in vivo. These data suggest that HepG2 and Hep3B are suitable models to study liver specific apolipoprotein gene expression.

A discoordinate increase in the cellular amount of 3-hydroxy-3-methylglutaryl-CoA reductase results in the loss of rate-limiting control over cholesterogenesis in a tumour cell-free system.

Cholesterol biosynthesis was characterized in cell-free post-mitochondrial supernatant systems prepared from both normal rat liver and Morris hepatoma 3924A. The rate of cholesterol synthesis per cell was 9-fold greater in the tumour system than in that from normal liver, and the tumour systems showed the loss of rate-limiting control at the hydroxymethylglutaryl-CoA reductase (HMGR)-catalysed step. The apparent absence of rate-limiting control over cell-free tumour cholesterogenesis was traced primarily to a discoordinate and dramatic increase in the amount of HMGR in the tumour relative to the liver system. Preliminary evidence for an altered control of the post-lanosterol portion of the pathway was also obtained with the tumour system.

Plasma lipoprotein(a) concentration is controlled by apolipoprotein(a) (apo(a)) protein size and the abundance of hepatic apo(a) mRNA in a cynomolgus monkey model.

The cynomolgus macaque was used as a model to study lipoprotein(a) (Lp(a)). Antibodies to Lp(a) were used in Ouchterlony and Western blot analysis to show that cynomolgus monkey and human Lp(a) were similar immunochemically. Monkey Lp(a) levels were measured by a quantitative sandwich enzyme-linked immunosorbent assay in 117 animals, and Lp(a) varied in concentration from 1 to 64 mg/dl. Individual monkeys had apo(a) glycoprotein sizes as either single- or double-band phenotypes that ranged from 400 to 750 kDa. Monkey apo(a) transcript lengths varied from 8.5 to 13.6 kilobases. The Lp(a) concentration, apo(a) glycoprotein size, and apo(a) transcript length distributions were similar to those in humans. In the monkeys, there was a very high correlation between apo(a) transcript size and apo(a) protein size (R = 0.93, p = 0.0001). This variation in apo(a) transcript and protein size was shown to be due to the number of kringle IV repeats in apo(a) mRNA and DNA. Monkey plasma Lp(a) concentrations correlated inversely with apo(a) glycoprotein size (R = 0.43, p = 0.0016) and directly with hepatic apo(a) mRNA abundance (R = 0.54, p = 0.004). Apo(a) transcript lengths did not correlate with hepatic apo(a) mRNA levels. This suggests that apo(a) size and mRNA levels have major independent effects on plasma Lp(a) concentration. In multivariate analysis, they account for up to 58% of the variability in Lp(a) concentration. In summary, these data provide insight into the regulation of Lp(a) levels and suggest that the cynomolgus monkey is a suitable model in which to study the role of Lp(a) in the pathogenesis of atherosclerosis.

Characterization of the mouse apolipoprotein Apoa-1/Apoc-3 gene locus: genomic, mRNA, and protein sequences with comparisons to other species.

In this report we present the genomic, cDNA, and predicted protein sequences for mouse apolipoproteins A-I and CIII, as well as sequence comparisons with other species. The genes for these apolipoproteins are within 2.5 kb of each other and convergently transcribed. The almost 9 kb of genomic sequence presented extends from 1298 bp 5' to the apolipoprotein A-I (Apoa-1) gene to 1249 bp 5' to the apolipoprotein CIII (Apoc-3) gene. The mouse Apoa-1 gene is 1.76 kb in length with four exons and three introns. The 5' flanking region contains TATA and CCAAT box sequences, an interferon responsive element homology, and potential binding sites for transcription factors CTF/NF1 and HNF4. Translation of the cDNA predicts that the mouse Apoa-1 primary transcript is 264 amino acids. The mouse Apoc-3 gene is 2.2 kb in length and also consists of four exons and three introns. The 5' flanking region contains TATA and CCAAT box sequences, RXR-1 and ARP-1 binding sites, and potential binding sites for transcription factors HNF4, NFkB, AP-1, and CTF/NF1. Translation of the cDNA predicts that the mouse Apoc-3 primary transcript is 99 amino acids. The clustering and genomic organization of the mouse Apoa-1 and Apoc-3 genes are similar to those of the rat and human genes. Significant sequence homologies between species exist for the proximal promoter and exonic regions of each gene, but not for the intronic or intergenic regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein(a) in diet-induced atherosclerosis in nonhuman primates.

Lipoprotein(a) (Lp[a]) is a low density lipoprotein particle that contains plasminogen-like apolipoprotein(a). Recent studies suggest an association of Lp(a) with atherosclerotic vascular disease. We have studied the accumulation of Lp(a) in atherosclerotic arteries of monkeys with diet-induced atherosclerosis. Immunohistochemistry with monospecific Lp(a) antisera revealed striking accumulations of Lp(a) in atherosclerotic coronary artery lesions. There was no Lp(a) in the normal, nonatherosclerotic arteries. Analysis of paired tissue and serum samples from 17 male hyperlipoproteinemic monkeys revealed a significant correlation between aortic wall Lp(a) and serum Lp(a) levels. The serum cholesterol level failed to correlate with either aortic Lp(a) or serum Lp(a). These results add further evidence for the potential role of Lp(a) in the pathogenesis of atherosclerosis.

Dietary fat elevates hepatic apoA-I production by increasing the fraction of apolipoprotein A-I mRNA in the translating pool.

Elevated plasma high density lipoprotein cholesterol (HDL-C) levels are associated with a decreased risk for coronary heart disease. Ironically, diets enriched in saturated fat and cholesterol (HF/HC diets), which tend to accelerate atherosclerotic processes by increasing LDL cholesterol levels, also raise HDL-C. We have recently reported, using a human apoA-I (hapoA-1) transgenic mouse model, that the elevation of HDL-C by a HF/HC diet is attributable, in part, to an increase in the hepatic production of hapoA-1. To further define the hepatocellular processes associated with this induction, we have prepared primary hepatocytes from hapoA-1 transgenic mice. Rates of hapoA-1 secretion were 40% greater from cells prepared from animals fed the HF/HC relative to a low fat-low cholesterol (LF/LC) control diet. The abundance of hapoA-1 mRNA in these cells was similar between hepatocytes prepared from the HF/HC and LF/LC diet fed animals, suggesting a post-transcriptional mechanism that does not involve mRNA stability. Inhibition of secretion using brefeldin A revealed an increase in cellular hapoA-1 accumulation. Thus, the HF/HC diet apparently affects hepatic hapoA-1 production via a mechanism that is manifest prior to the exit of newly synthesized hapoA-1 from the Golgi. Pulse-chase experiments revealed a 39% greater peak hapoA-1 synthesis, with no difference in the degradation of total labeled hapoA-1 protein, as a result of the HF/HC diet feeding. Finally, resolution of liver S10 extracts via sucrose density sedimentation and metrizamide density equilibrium gradient centrifugation analyses both revealed similar increases (31 and 24%, respectively) in the relative percentage of hapoA-1 mRNA associated with the translating polysomal fractions as a result of the HF/HC feeding. Together, these data suggest that the HF/HC diet affects hepatic hapoA-1 production via a specific modulation in the relative amount of hapoA-1 mRNA in the polysomal pool. These observations provide an opportunity to explore a new mechanism regulating apoA-1 production and might lead to the development of novel therapies to elevate plasma HDL-C levels.

Probucol decreases apolipoprotein A-I transport rate and increases high density lipoprotein cholesteryl ester fractional catabolic rate in control and human apolipoprotein A-I transgenic mice.

Probucol effects on lipoprotein metabolism were determined in control and human apolipoprotein A-I transgenic (HuAITg) mice. In control mice, probucol reduced total cholesterol from 67 +/- 2 to 25 +/- 2 mg/dl by reducing high density lipoprotein (HDL) cholesterol from 46 +/- 20 to 14 +/- 1 mg/dl and low density lipoprotein (LDL) cholesterol from 11 +/- 1 to 5 +/- 1 mg/dl. Apolipoprotein (apo) A-I levels were reduced from 122 +/- 8 to 56 +/- 5 mg/dl. In HuAITg mice, probucol reduced total cholesterol from 121 +/- 5 to 77 +/- 3 mg/dl by reducing HDL cholesterol from 84 +/- 4 to 56 +/- 3 mg/dl and LDL cholesterol from 19 +/- 2 to 11 +/- 2 mg/dl. Human apo A-I levels were reduced from 267 +/- 13 to 144 +/- 12 mg/dl and mouse apo A-I levels from 18 +/- 2 to 9 +/- 2 mg/dl. Control animals have primarily a monodisperse HDL with a particle diameter of 10 nm. Probucol did not appear to change the particle size distribution in the control animals. The HuAITg mice have a polydisperse HDL with particle diameters of 10.1 and 8.5 nm. Probucol treatment of these animals resulted in HDL with particle diameters of 9.4 and 8.5 nm, apparently reducing the size of the larger particles. In vivo turnover studies revealed that the reduction in apo A-I was primarily due to a decrease in transport rate, whereas the reduction in HDL cholesterol was primarily due to an increase in HDL cholesteryl ester fractional catabolic rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal expression of the human apoA-I gene in transgenic mice is controlled by a DNA region 3' to the gene in the promoter of the adjacent convergently transcribed apoC-III gene.

The apoA-I gene in humans is principally expressed in liver and small intestine. Using transgenic mice, we previously showed that 256 bp of 5' flanking DNA was sufficient for liver expression, but as much as 5.5 kb of 5' and 4.0 kb of 3' DNA did not allow intestinal expression of the human apoA-I transgene. In the current study, a 10.5 kb DNA construction containing the apoA-I and the adjacent convergently transcribed apoC-III genes, which extends from 300 bp 5' to the apoA-I gene to 2.5 kb 5' to the apoC-III gene, produced high levels of apoA-I intestinal expression. A similar DNA construction ending 1.4 kb 5' to the apoC-III gene also allowed apoA-I intestinal expression. The DNA region from 0.2 to 1.4 kb 5' to the apoC-III gene was then cloned 1.7 kb 3' to the apoA-I gene in both orientations in the absence of apoC-III gene sequences. Intestinal apoA-I expression was also achieved with both of these constructions. In summary, these in vivo experiments suggest that the intestinal control region for the apoA-I gene is distinct from the liver control region, resides 3' to the gene in the promoter of the adjacent apoC-III gene, and has some properties of a tissue-specific enhancer.

Reduction of lipoprotein(a) following treatment with lovastatin in patients with unremitting nephrotic syndrome.

Pharmocologic treatment of the hyperlipidemia associated with the nephrotic syndrome with lovastatin has been previously shown to be safe and effective. However, there is no information on the effect of lovastatin treatment on plasma lipoprotein(a) [Lp(a)] levels in patients with the nephrotic syndrome. We administered lovastatin (40 to 80 mg/day) to 20 adult patients with unremitting nephrotic syndrome for 8 weeks to assess its effect on plasma Lp(a) and other plasma lipid concentrations. Apoprotein(a) (apo(a)) phenotype was determined in all patients. Patients were grouped according to their plasma Lp(a) levels. Those with elevated plasma Lp(a) (> or = 30 mg/dL) were placed in group I and those with normal Lp(a) levels (< 30 mg/dL) were placed in group II. Mean total cholesterol and LDL cholesterol were similarly and significantly reduced in groups I and II (-35.9% and -43.3%, P < 0.0005, P < 0.0005 group I, and -31.0% and -42.0%, P < 0.02, P < 0.03 group II, respectively). The median reduction in plasma Lp(a) was -32% (P < 0.003) in nephrotic patients in group I, whereas the median decline in plasma Lp(a) levels in nephrotic patients in group II was only -8.0% (P = 0.052). The overall frequency of the high molecular weight (M(r)) apo(a) phenotype S4 was 70% in nephrotic patients. There was no correlation between plasma Lp(a) and apo(a) phenotype. Treatment with lovastatin results in a favorable response in terms of total and low-density lipoprotein cholesterol lowering in patients with the nephrotic syndrome; however, plasma Lp(a) levels are uniformly and significantly reduced only in nephrotic patients with elevated baseline plasma Lp(a) concentrations. There was no correlation between plasma Lp(a) concentration and other lipid and biochemical parameters.

ApoA-I knockout mice: characterization of HDL metabolism in homozygotes and identification of a post-RNA mechanism of apoA-I up-regulation in heterozygotes.

The major high density lipoprotein (HDL) apolipoprotein, apoA-I, was knocked out by gene targeting in ES cells to provide a model for the study of HDL metabolism and its relationship to plasma and tissue cholesterol metabolism. HDL and non-HDL cholesterol (HDL-C) were reduced in apoA-I-deficient mice. Feeding a high fat-high cholesterol diet raised HDL-C minimally in apoA-I knockout compared to the large increase seen in control mice, suggesting an interaction between diet and apoA-I genotype. In apoA-I-deficient mice, HDL was normal in size but altered in composition. Compared to control mice there was more triglyceride and free cholesterol and less cholesteryl ester (CE), suggesting that apoA-I-deficient HDL is a poor substrate for hepatic lipase and lecithin:cholesterol acyltransferase (LCAT). The metabolic basis of the low HDL-C levels in the apoA-I knockout mice was decreased flux into the HDL CE pool. The absolute delivery of HDL CE to both peripheral tissues and liver was also decreased. As tissue cholesterol levels and synthesis were unchanged, the decreased flux of cholesterol into the HDL CE pool was most likely due to decreased efflux of cholesterol from the peripheral tissues and decreased functional LCAT activity. The low HDL-C state in the apoA-I-deficient mouse was associated with an absolute decrease in unidirectional transport of cholesterol from peripheral tissues to the liver but this did not lead to cholesterol accumulation in the periphery or a cholesterol deficit in the liver; nor was there altered peripheral tissue HMG-CoA reductase activity. The only sign of decreased cholesterol flux to the liver was a 2.3-fold decrease in liver cholesterol 7 alpha-hydroxylase mRNA, suggesting decreased bile acid synthesis. In the apoA-I knockout mouse model it appears that low HDL levels create a new steady state in which decreased cholesterol is delivered to both peripheral tissues and the liver.

Human placental tissue expresses a novel 22.7 kDa apolipoprotein A-I-like protein.

Since apolipoprotein A-I (apo A-I) and HDL stimulate the expression of the placental hormone human placental lactogen (hPL), experiments were performed to determine whether the human placenta synthesizes apo A-I. Western blot analysis of a partially purified extract of human term placenta with an antiserum to human apo A-I yielded an immunoreactive band with an apparent mass of approximately 23.5 kDa, which is smaller than human plasma apo A-I (28 kDa). HPLC chromatography of the partially purified placental extract on a preparative reverse-phase C-18 column yielded two fractions that reacted to the apo A-I antiserum. The mass of both fractions by mass spectral analysis was 22 721 daltons, and N-terminal amino acid sequences were identical to the first four amino acids of apo A-I (Asp, Glu, Pro, Pro). The apo A-I-like protein was not a proteolytic product of apo A-I since Northern analysis of placental RNA with a 641 bp apo A-I cDNA fragment encoding most of the 5' region of the apo A-I mRNA detected a single band of 850 nt, which is smaller than the size of apo A-I mRNA (1100 nt). Placental mRNA, however, did not hybridize with a 3' apo A-I riboprobe, indicating that the 3' region of the apo A-I-like mRNA is different from that of apo A-I mRNA. Differences in the mRNAs were confirmed by S1 nuclease analysis of placental RNA with a cDNA probe that included the 3' end of the apo A-I cDNA and by RT-PCR analysis with a series of oligonucleotide primers that span the entire cDNA for apo A-I. Since there is only a single apo A-I gene in the human genome, these findings strongly suggest that human placental tissue expresses a novel 22.7 kDa apo A-I-like protein (ALP) that results from alternative splicing of the apo A-I primary transcript.

Cyclosporin A has divergent effects on plasma LDL cholesterol (LDL-C) and lipoprotein(a) [Lp(a)] levels in renal transplant recipients. Evidence for renal involvement in the maintenance of LDL-C and the elevation of Lp(a) concentrations in hemodialysis patients.

Cardiovascular disease is the major cause of mortality in renal transplant recipients. Plasma levels of low-density lipoprotein cholesterol (LDL-C) are often elevated following renal transplantation, and the immunosuppressant cyclosporin A has been implicated as a predisposing factor for posttransplantation hyperlipidemia. Lipoprotein(a) [Lp(a)] is an LDL-like lipoprotein particle; elevated levels of Lp(a) provide an independent and significant risk factor for cardiovascular disease. Plasma concentrations of Lp(a) vary greatly among individuals, and the mechanisms that govern changes in their levels in transplant patients are unknown. The effect(s) of cyclosporin A on Lp(a) was studied in two groups of renal transplantation patients. In group I plasma lipoproteins including Lp(a) were measured before and after successful renal transplantation; this group received both prednisone and cyclosporin A for immunosuppression. Group II patients were studied after renal transplantation and received prednisone alone for immunosuppression. Following surgery, group I patients demonstrated increased plasma concentrations of LDL-C (mean +/- SEM range, 111 +/- 6 to 142 +/- 17 mg/dL; P < .005). In contrast, plasma Lp(a) levels for this group were markedly decreased after renal transplantation (median, 34.3 to 19.7 mg/dL). Patients not treated with cyclosporin A (group II) exhibited mean LDL-C and median Lp(a) levels (118 +/- 42 and 33.1 mg/dL, respectively) that were remarkably similar to those observed before renal transplantation (group I). These data confirm that hyperlipidemia following renal transplantation is associated with cyclosporin A therapy and show that this drug has opposing effects on plasma Lp(a) and LDL-C accumulations.(ABSTRACT TRUNCATED AT 250 WORDS)