Richard M. Lawn

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.

Lipoprotein(a) and atherosclerosis.

Lipoprotein(a) [Lp(a)], a lipoprotein variant, was relegated for almost 25 years to the study of a few specialists. During the past 3 to 4 years, however, there has been a tremendous upsurge of interest in Lp(a), primarily because of multidisciplinary efforts in structural and molecular biology. Findings emerging from these efforts include the following: Lp(a) represents a cholesteryl-ester, low-density-lipoprotein (LDL)-like particle with apolipoprotein (apo) B-100 linked to apo(a); apo(a) is a glycoprotein coded by a single gene locus on the long arm of chromosome 6, which has several alleles, accounting for its remarkable size polymorphism (300 to 800 kD); apo(a) size polymorphism relates to plasma levels and density distribution of Lp(a); apo(a) is strikingly similar to plasminogen; and in vitro, Lp(a), in appropriate levels, competes for some physiologic functions of plasminogen in the coagulation and fibrinolytic cascade and may thus be thrombogenic. The LDL-like properties of Lp(a) may also confer atherogenic potential, but the mechanisms underlying this atherogenicity remain to be defined. In epidemiologic studies, high plasma Lp(a) levels have been associated with an increased incidence of atherosclerotic cardiovascular disease, especially in patients less than 60 years of age. Moreover, Lp(a) has been found as an intact particle in the arterial intima, particularly in association with atherosclerotic plaque. This finding suggests that Lp(a) can transverse the endothelium, possibly by a non-receptor-mediated process, and, at the intimal level, acquire thrombogenic and atherogenic potentials. Current information justifies the need to determine plasma Lp(a) levels in patients with a history of atherosclerotic cardiovascular disease. Unfortunately, the available techniques need to be standardized. Apolipoprotein(a) exists in isoforms of different sizes, and the importance of determining apo(a) phenotypes in clinical practice remains to be established.

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.

Apolipoprotein(a) Gene Enhancer Resides within a LINE Element

Apolipoprotein(a), (apo(a)), is the distinguishing protein portion of the lipoprotein(a) particle, elevated plasma levels of which are a major risk factor for cardiovascular disease. A search for enhancer elements that control the transcription of the apo(a) gene led to the identification of an upstream element that contains target binding sites for members of the Ets and Sp1 nuclear protein families. The enhancer element functions in either orientation to confer a greater than 10-fold increase in the activity of the apo(a) minimal promoter in cultured hepatocyte cells. Unexpectedly, the enhancer element is located within a LINE retrotransposon element, suggesting that LINE elements may function as mobile regulatory elements to control the expression of nearby genes.

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.

Liver X receptors α and β regulate renin expression in vivo

The renin-angiotensin-aldosterone system controls blood pressure and salt-volume homeostasis. Renin, which is the first enzymatic step of the cascade, is critically regulated at the transcriptional level. In the present study, we investigated the role of liver X receptor alpha (LXR(alpha)) and LXR(beta) in the regulation of renin. In vitro, both LXRs could bind to a noncanonical responsive element in the renin promoter and regulated renin transcription. While LXR(alpha) functioned as a cAMP-activated factor, LXR(beta) was inversely affected by cAMP. In vivo, LXRs colocalized in juxtaglomerular cells, in which LXR(alpha) was specifically enriched, and interacted with the renin promoter. In mouse models, renin-angiotensin activation was associated with increased binding of LXR(alpha) to the responsive element. Moreover, acute administration of LXR agonists was followed by upregulation of renin transcription. In LXR(alpha) mice, the elevation of renin triggered by adrenergic stimulation was abolished. Untreated LXR(beta) mice exhibited reduced kidney renin mRNA levels compared with controls. LXR(alpha)LXR(beta) mice showed a combined phenotype of lower basal renin and blunted adrenergic response. In conclusion, we show herein that LXR(alpha) and LXR(beta) regulate renin expression in vivo by directly interacting with the renin promoter and that the cAMP/LXR(alpha) signaling pathway is required for the adrenergic control of the renin-angiotensin system.

THE RECURRING EVOLUTION OF LIPOPROTEIN(A) : INSIGHTS FROM CLONING OF HEDGEHOG APOLIPOPROTEIN(A)

The lipoprotein Lp(a), a major inherited risk factor for atherosclerosis, consists of a low density lipoprotein-like particle containing apolipoprotein B-100 plus the distinguishing component apolipoprotein(a) (apo(a)). Human apo(a) contains highly repeated domains related to plasminogen kringle four plus single kringle five and protease-like domains. Apo(a) is virtually confined to primates, and the gene may have arisen during primate evolution. One exception is the occurrence of an Lp(a)-like particle in the hedgehog. Cloning of the hedgehog apo(a)-like gene shows that it is distinctive in form and evolutionary history from human apo(a), but that it has acquired several common features. It appears that the primate and hedgehog apo(a) genes evolved independently by duplication and modification of different domains of the plasminogen gene, providing a novel type of “convergent” molecular evolution.

Impairment of Collateral Formation in Lipoprotein(a) Transgenic Mice Therapeutic Angiogenesis Induced by Human Hepatocyte Growth Factor Gene

Background—Although lipoprotein(a) (Lp[a]) is a risk factor for atherosclerosis, no study has documented the effects of Lp(a) on angiogenesis. In this study, we examined collateral formation in peripheral arterial disease (PAD) model in Lp(a) transgenic mice. In addition, we examined the feasibility of gene therapy by using an angiogenic growth factor, hepatocyte growth factor (HGF), to treat PAD in the presence of high Lp(a). Methods and Results—In Lp(a) transgenic mice, the degree of natural recovery of blood flow after operation was significantly lower than that in nontransgenic mice. Of importance, there was a significant negative correlation between serum Lp(a) concentration and the degree of natural recovery of blood flow (P <0.05). In addition, Lp(a) significantly stimulated the growth of vascular smooth muscle, accompanied by the phosphorylation of ERK. These data demonstrated the association of impairment of collateral formation with serum Lp(a) concentration. Thus, we examined the feasibility of therapeutic angiogenesis by using HGF, with the goal of progression to human gene therapy. Intramuscular injection of HGF plasmid resulted in a significant increase in blood flow even in Lp(a) transgenic mice, accompanied by the detection of human HGF protein. A significant increase in capillary density also was detected in Lp(a) transgenic mice transfected with human HGF compared with control (P <0.01). Conclusions—Overall, a high serum Lp(a) concentration impaired collateral formation. Although the delay of angiogenesis in high serum Lp(a) might diminish angiogenesis, intramuscular injection of HGF plasmid induced therapeutic angiogenesis in the Lp(a) transgenic ischemic hindlimb mouse model as potential therapy for PAD.

Phosphatidylcholine Hydrolysis Is Required for Pancreatic Cholesterol Esterase- and Phospholipase A2-facilitated Cholesterol Uptake into Intestinal Caco-2 Cells

Pancreatic secretion is required for efficient cholesterol absorption by the intestine, but the factors responsible for this effect have not been clearly defined. To identify factors involved and to investigate their role in cholesterol uptake, we studied the effect of Viokase®, a porcine pancreatic extract, on cholesterol uptake into human intestinal Caco-2 cells. Viokase is capable of facilitating cholesterol uptake into these cells such that the level of uptake is 5-fold higher in the presence of solubilized Viokase. This stimulation is time-dependent and is dependent on the presence of bile salt. However, bile salt-stimulated pancreatic cholesterol esterase, which has been proposed to mediate cholesterol uptake, is not fully responsible. The major cholesterol transport activity was purified and identified as pancreatic phospholipase A2. Anti-phospholipase A2 antibodies abolished virtually all of the phospholipase A2 and cholesterol transport activity of solubilized Viokase. We demonstrate that both phospholipase A2 and cholesterol esterase increase cholesterol uptake by hydrolyzing the phosphatidylcholine that is used to prepare the cholesterol-containing micelles. In the absence of cholesterol esterase or phospholipase A2, uptake of cholesterol from micelles containing phosphatidylcholine is not as efficient as uptake from micelles containing phospholipase A2-hydrolytic products. These results indicate that phospholipase A2 may mediate cholesterol absorption by altering the physical-chemical state of cholesterol within the intestine.

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.