Antifibrinolytic activity of apolipoprotein(a) in vivo: human apolipoprotein(a) transgenic mice are resistant to tissue plasminogen activator-mediated thrombolysis.

The extensive homology between apolipoprotein(a) and plasminogen has led to the hypothesis that the increased risk for atherosclerosis, cardiac disease and stroke associated with elevated levels of apolipoprotein(a) may reflect modulation of fibrinolysis. We have investigated the role of apolipoprotein(a) on clot lysis in transgenic mice expressing the human apolipoprotein(a) gene. These mice develop fatty streak lesions resembling early lesions of human atherosclerosis. Pulmonary emboli were generated in mice by injection, through the right jugular vein, of a human platelet-rich plasma clot radiolabelled with technetium-99m-labelled antifibrin antibodies. Tissue plasminogen activator was introduced continuously via the right jugular vein. Clot lysis, determined by ex vivo imaging, was depressed in mice carrying the apolipoprotein(a) transgene relative to their sex-matched normal littermates. These results directly demonstrate an in vivo effect of apolipoprotein(a) on fibrinolysis, an effect that may contribute to the pathology associated with elevated levels of this protein.

The serum concentration of active transforming growth factor-beta is severely depressed in advanced atherosclerosis.

Recent evidence has led us to propose that transforming growth factor-beta (TGF-beta) is a key inhibitor of atherosclerosis. We show here that a population of patients with advanced atherosclerosis all have less active TGF-beta in their sera than patients with normal coronary arteries, with a fivefold difference in average concentration between the two groups. This correlation with atherosclerosis is much stronger than for other known major risk factors and it may therefore have important diagnostic and prognostic significance. Aspirin medication correlates with an increase in active TGF-beta concentration, indicating that therapeutic interventions for TGF-beta are possible.

Identification of a missense mutation in the factor VIII gene of a mild hemophiliac.

DNA probes derived from the cloned factor VIII gene can be used to detect mutations in the factor VIII gene of hemophiliacs. DNA hybridization analysis led to the identification of two contrasting point mutations in the same codon. In a severe hemophiliac with no detectable factor VIII activity, the normal arginine codon (number 2307) is converted to a stop codon, while in a mild hemophiliac with 10 percent of normal activity, this same codon is converted to glutamine.

DNA sequence of two closely linked human leukocyte interferon genes.

A single recombinant lambda bacteriophage isolated from a human genome library contains two closely related human interferon genes of the leukocyte or alpha type. The two genes are separated by 12 kilobase pairs and are oriented in the same direction with respect to transcription. Comparisons of the DNA sequences of these two genes and interferon complementary DNA clones indicate that the two interferon genes lack intervening sequences.

Proliferation of human smooth muscle cells promoted by lipoprotein(a).

Elevated blood concentrations of lipoprotein(a) [Lp(a)] and its constituent, apolipoprotein(a) [apo(a)], constitute a major risk factor for atherosclerosis, but their physiological activities remain obscure. Lp(a) and purified apo(a) stimulated the growth of human smooth muscle cells in culture. This effect resulted from inhibition of plasminogen activation, and consequently the activation by plasmin of latent transforming growth factor-beta, which is an inhibitor of smooth muscle cell growth. Because smooth muscle proliferation is one of the hallmarks of atherosclerotic lesions, these results point to a plausible mechanism for the atherogenic activity of Lp(a).

Human lipoprotein lipase complementary DNA sequence.

Lipoprotein lipase is a key enzyme of lipid metabolism that acts to hydrolyze triglycerides, providing free fatty acids for cells and affecting the maturation of circulating lipoproteins. It has been proposed that the enzyme plays a role in the development of obesity and atherosclerosis. The human enzyme has been difficult to purify and its protein sequence was heretofore undetermined. A complementary DNA for human lipoprotein lipase that codes for a mature protein of 448 amino acids has now been cloned and sequenced. Analysis of the sequence indicates that human lipoprotein lipase, hepatic lipase, and pancreatic lipase are members of a gene family. Two distinct species of lipoprotein lipase messenger RNA that arise from alternative sites of 3'-terminal polyadenylation were detected in several different tissues.

Clustering of leukocyte and fibroblast interferon genes of human chromosome 9.

At least ten leukocyte interferon genes and the single known fibroblast interferon gene have been localized on the pter leads to q12 region of human chromosome 9. Gene mapping was accomplished by blot hybridization of cloned interferon complementary DNA to DNA from human-mouse cell hybrids with a translocation involving human chromosome 9. Supporting evidence suggests these genes are clustered.

Lipoprotein(a) vascular accumulation in mice. In vivo analysis of the role of lysine binding sites using recombinant adenovirus.

Although the mechanism by which lipoprotein(a) [Lp(a)] contributes to vascular disease remains unclear, consequences of its binding to the vessel surface are commonly cited in postulated atherogenic pathways. Because of the presence of plasminogen-like lysine binding sites (LBS) in apo(a), fibrin binding has been proposed to play an important role in Lp(a)'s vascular accumulation. Indeed, LBS are known to facilitate Lp(a) fibrin binding in vitro. To examine the importance of apo(a) LBS in Lp(a) vascular accumulation in vivo, we generated three different apo(a) cDNAs: (a) mini apo(a), based on wild-type human apo(a); (b) mini apo(a) containing a naturally occurring LBS defect associated with a point mutation in kringle 4-10; and (c) human- rhesus monkey chimeric mini apo(a), which contains the same LBS defect in the context of several additional changes. Recombinant adenovirus vectors were constructed with the various apo(a) cDNAs and injected into human apoB transgenic mice. At the viral dosage used in these experiments, all three forms of apo(a) were found exclusively within the lipoprotein fractions, and peak Lp(a) plasma levels were nearly identical (approximately 45 mg/dl). In vitro analysis of Lp(a) isolated from the various groups of mice confirmed that putative LBS defective apo(a) yielded Lp(a) unable to bind lysine-Sepharose. Quantitation of in vivo Lp(a) vascular accumulation in mice treated with the various adenovirus vectors revealed significantly less accumulation of both types of LBS defective Lp(a), relative to wild-type Lp(a). These results indicate a correlation between lysine binding properties of Lp(a) and vascular accumulation, supporting the postulated role of apo(a) LBS in this potentially atherogenic characteristic of Lp(a).

Interaction of recombinant apolipoprotein(a) and lipoprotein(a) with macrophages.

Elevated plasma levels of lipoprotein(a), Lp(a), represent a major, inherited risk factor for coronary heart disease, although the mechanism of its action remains unknown. Lp(a) is distinguished from the related LDL particle by the addition of apolipoprotein(a), apo(a). The presence of this large glycoprotein is likely to affect the binding of the particle to the LDL receptor and/or other receptors which may contribute to the atherogenic potential of Lp(a). Here we demonstrate the binding to macrophages of Lp(a) and pure recombinant apo(a) protein, via a specific, high-affinity receptor. This binding could lead to foam cell formation and the localization of Lp(a) to atherosclerotic plaques.

Modification of apolipoprotein(a) lysine binding site reduces atherosclerosis in transgenic mice.

Lipoprotein(a) contributes to the development of atherosclerosis through the binding of its plasminogen-like apolipoprotein(a) component to fibrin and other plasminogen substrates. Apolipoprotein(a) contains a major lysine binding site in one of its kringle domains. Destruction of this site by mutagenesis greatly reduces the binding of apolipoprotein(a) to lysine and fibrin. Transgenic mice expressing this mutant form of apolipoprotein(a) as well as mice expressing wild-type apolipoprotein(a) have been created in an inbred mouse strain. The wild-type apolipoprotein(a) transgenic mice have a fivefold increase in the development of lipid lesions, as well as a large increase in the focal deposition of apolipoprotein(a) in the aorta, compared with the lysine binding site mutant strain and to nontransgenic littermates. The results demonstrate the key role of this lysine binding site in the pathogenic activity of apolipoprotein(a) in a murine model system.

The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway.

The ABC1 transporter was identified as the defect in Tangier disease by a combined strategy of gene expression microarray analysis, genetic mapping, and biochemical studies. Patients with Tangier disease have a defect in cellular cholesterol removal, which results in near zero plasma levels of HDL and in massive tissue deposition of cholesteryl esters. Blocking the expression or activity of ABC1 reduces apolipoprotein-mediated lipid efflux from cultured cells, and increasing expression of ABC1 enhances it. ABC1 expression is induced by cholesterol loading and cAMP treatment and is reduced upon subsequent cholesterol removal by apolipoproteins. The protein is incorporated into the plasma membrane in proportion to its level of expression. Different mutations were detected in the ABC1 gene of 3 unrelated patients. Thus, ABC1 has the properties of a key protein in the cellular lipid removal pathway, as emphasized by the consequences of its defect in patients with Tangier disease.

Localization of human ATP-binding cassette transporter 1 (ABC1) in normal and atherosclerotic tissues.

The present study examines the expression of ATP-binding cassette transporter 1 (ABC1) mRNA in normal and atherosclerotic tissues by using in situ hybridization in an effort to better understand the function of this cholesterol transport protein. Samples of normal baboon tissues as well as human normal and atherosclerotic aortas were hybridized with (35)S-labeled ABC1 sense and antisense riboprobes. Widespread expression of ABC1 was observed generally in tissues containing inflammatory cells and lymphocytes. Other noninflammatory cells that were also sites of ABC1 synthesis included the ductal cells of the kidney medulla, Leydig cells in the testis, and glial cells in the baboon cerebellum. Although normal veins and arteries did not express ABC1 mRNA, it was found to be upregulated in the setting of atherosclerosis, where widespread expression was found in macrophages within atherosclerotic lesions. These results are consistent with the proposed role of ABC1 in cholesterol transport in inflammatory cells. The specific upregulation of ABC1 mRNA in the setting of atherosclerosis probably reflects the response of leukocytes to cholesterol loading. However, the presence of ABC1 in ductal cells of the kidney medulla and in the small intestine suggest a more general role for this protein in cholesterol transport in other cell types.

Determination of temporal expression patterns for multiple genes in the rat carotid artery injury model.

Vascular injury induces extensive alteration to the extracellular matrix (ECM). These changes contribute to lesion formation and promote cell migration and proliferation. To elucidate ECM response to arterial injury, we used real-time polymerase chain reaction monitoring to quantitate the expression levels of 81 genes involved in the synthesis and breakdown of ECM as well as receptors and signaling proteins that communicate and respond to ECM molecules. The temporal regulation of gene expression in the carotid was measured at 1, 3, 5, 7, 9, 14, and 28 days postinjury. Among the 68 genes that showed detectable expression by our method, 47 (69%) were significantly induced or repressed over time, confirming the extensive ECM gene response in this model. More ECM-related genes (31) were regulated at day 1 than at any other time point, and the number of regulated genes decreased over time. However, 14 of the genes were still induced or repressed at day 28, indicating that return to preinjury expression patterns did not occur and no new steady state was achieved over 28 days. In spite of the large number of changes in gene expression, only a small number of expression patterns was observed, suggesting that ECM-related genes could potentially be coregulated.

Lipoprotein(a) and atherogenesis.

Lipoprotein(a) is a major inherited risk factor for atherosclerosis. Many of its activities depend on its plasminlike component, apolipoprotein(a). In vitro studies suggest that apolipoprotein(a) could enhance lipid deposition through binding to sites in the vessel wall, interfere with fibrinolysis, and modulate smooth muscle cell activity. Human and animal studies will be necessary to establish the physiologic importance of these mechanisms.

Characterization of the mouse pancreatic/mammary gland cholesterol esterase-encoding cDNA and gene.

Pancreatic cholesterol esterase (CEase) is involved in cholesterol ester hydrolysis and transport of cholesterol into intestinal cells. Isolation and sequencing of the mouse CEase cDNA indicates that the murine enzyme is very similar to the human enzyme, with the exception of the highly repeated C-terminal Pro-rich repeats. Reverse transcription-polymerase chain reaction analysis reveals that the message is synthesized in the pancreas of mice, but not in the liver.

Histopathology of arterial lesions in LPA transgenic mice on cholesterol-enriched chow.

The aortic root from 21 LPA transgenic mice and 18 control litter mates on cholesterol enriched chow were studied histologically for the presence of atherosclerotic lesions. Serial sections were cut and the total area of the lesions was measured by use of computerised image analysis. Lipid staining lesions were found in 17 aortas of the transgenic mice and were five times more common than in the controls. Foam cell lesions were the only type of lesion in 12 of the aortas from transgenic animals, while five animals had developed fibrofatty lesions. Immunostaining revealed monocytes/macrophages on the endothelial surface, and in the subendothelial space of foam cell lesions. In fibrofatty lesions, spindle shaped cells formed a cap around the lipid core. This study supports the view that transgenic mice expressing human apolipoprotein (a) on a high fat and cholesterol diet, are more susceptible to aortic lesions than control mice and develop early atherosclerotic lesions comparable to lesions in man. Aminoguanidin in the drinking water had no effect on the aortic lesions, but lesion size was significantly, negatively correlated with plasma glucose concentration.

Endothelium-dependent vasorelaxation in the aorta of transgenic mice expressing human apolipoprotein(a) or lipoprotein(a).

Elevated plasma level of lipoprotein(a) (Lp(a)) is a well established risk factor for premature atherosclerosis and coronary artery disease. Recent studies showed impaired endothelium-dependent vasodilatation in humans with elevated plasma Lp(a). However, these human studies could not determine whether (1) elevated Lp(a) levels alone are the cause of endothelial dysfunction (these patients had multiple risk factors), and (2) native or oxidatively modified Lp(a) contributes to endothelial dysfunction (no measurements of native/oxidized Lp(a) ratio was reported in humans). In order to test whether apo(a) (an essential component of Lp(a) which is required for binding to endothelial cells) and native Lp(a) cause endothelial dysfunction, in the present study we tested endothelium-dependent vasorelaxation in aortic rings isolated from control and transgenic male mice either expressing the human apo(a) gene (TgA) or both the human apo(a) and human apo B100 genes (TgL). The TgA mice had plasma apo(a) levels of 8.8 +/- 1.2 mg/dl (n=6) and the double transgenic TgL mice had plasma Lp(a) levels of 15.3 +/- 1.4 mg/dl (n=8). Isolated aortic rings with and without endothelium were mounted in organ chambers and contracted with U46619 (10(-8) M) in the presence of ibuprofen (10(-5) M). Acetylcholine caused concentration-dependent (10(-9)-10(-5) M) relaxation, which could be prevented by endothelium removal and by NG-L-nitro-arginine (10(-4) M). Basal and acetylcholine-stimulated endothelium-dependent relaxation and endothelium-independent relaxation to nitroglycerin (10(-6) M) were not significantly different in aortic rings isolated from control and TgA or TgL mice. Twenty-four hour incubation of aortic rings isolated from control mice with recombinant human apo(a) or native Lp(a) (up to 300 microg/ml) caused no impairment of endothelium-dependent relaxations. In contrast, incubation with oxidized Lp(a) (50 microg/ml) or oxidized LDL (250 microg/ml) caused significant suppression of acetylcholine-induced endothelium-dependent vasorelaxation. These results show for the first time that elevated plasma levels of apo(a) and Lp(a) do not cause endothelial dysfunction in transgenic mice.

Cloning and expression of human tissue factor cDNA.

Tissue factor is a membrane protein that plays an essential role in the initiation of blood coagulation. When exposed to the circulation, tissue factor interacts with the serine protease factor VII, and the complex triggers fibrin clot formation by activating both factors IX and X of the coagulation cascade. This report describes the cloning and expression of the complementary DNA (cDNA) for human tissue factor. The cDNA encodes a protein of 263 amino acids preceded by a 32 amino acid signal peptide. The predicted protein sequence contains a potential hydrophobic membrane anchoring domain at its carboxy terminus, and bears no significant homology to any other known protein. Tissue factor mRNA of 2400 nucleotides was detected in adipose, adrenal, small intestine and a number of other tissues by Northern blot hybridization analysis. In order to confirm the identity of the cDNA, an expression vector containing the cloned cDNA was used to transfect cultured mammalian cells. These cells produced active tissue factor which was assayed using purified factors VII and X.

The apolipoprotein(a) gene: characterization of 5' flanking regions and expression in transgenic mice.

The human genome contains at least six homologues of the apolipoprotein(a) and plasminogen genes, which contain over 90% identity in the region corresponding to 5' flanking sequences and exon one. This region of the apolipoprotein(a) gene contains transcription control elements, and sequence differences in this region account for some of the variation in plasma Lp(a) concentration. Transgenic mice expressing human apolipoprotein(a) develop aortic lesions resembling the early stages of human atherosclerosis after 3.5 months on a high fat diet. The transgenic mouse model should prove a useful system for studying the role of apolipoprotein(a) in disease.

Electron-microscopic and hydrodynamic characterization of recombinant apolipoprotein (a) and its association with LDL.

A recombinant apo(a) containing 17 kringle 4 domains as well as the kringle 5 and protease domains of apo(a) was characterized by hydrodynamic studies and electron microscopy. Recombinant apo(a) is a monomer in solution with a molecular weight of 325,000 by sedimentation equilibrium and 320,000 by sedimentation and diffusion, and it is a highly asymmetric molecule with a frictional ratio of 2.2. In the electron microscope recombinant apo(a) is visualized as a flexible chain of domains approximately 800 A long. Sedimentation velocity studies also demonstrate that when it is mixed with LDL, recombinant apo(a) reversibly forms an Lp(a)-like complex with a 1:1 stoichiometry; moreover, complex formation is inhibited by 6-amino hexanoic acid. Hydrodynamic modeling and electron microscopy suggest that only a small portion of the r-apo(a) molecule interacts with the LDL and the rest of the chain extends into solution. Preliminary studies indicate that recombinant apo(a) also binds mouse LDL.