Overexpression Of apoE Research Articles

Effect of macrophage ApoE on atherosclerosis in LDL-receptor deficient mice

Apolipoprotein E (ApoE) plays an important role in the development of atherosclerosis. Previous studies provide evidence for an atheroprotective role of ApoE in mouse models on the ApoE deficient (ApoE−/−) background. However, it is not clear whether this is also true on the LDL-receptor deficient (LDLR−/−) background. Transgenic mice carrying hApoE coding sequences in a chicken lysozyme expression cassette were generated. Transgene expression was directed into macrophages, expressing low levels of hApoE. Expression of the hApoE transgene was not sufficient to correct hypercholesterolemia. However, lesion area at the brachiocephalic artery (BCA) was significantly reduced (−72%) in female hApoE transgenic mice on the LDLR−/− background. This was associated with increased cholesterol efflux in macrophages of transgenic animals on the ApoE−/− background. We conclude that over-expression of ApoE in macrophages might be useful as a therapeutic principle for the prevention of atherosclerosis.

A Dominant Negative Form of the Transcription Factor c-Jun Affects Genes That Have Opposing Effects on Lipid Homeostasis in Mice

c-Jun is a transcription factor activated by phosphorylation by the stress-activated protein kinase/c-Jun N-terminal kinase pathway in response to extracellular signals and cytokines. We show that adenovirus-mediated gene transfer of the dominant negative form of c-Jun (dn-c-Jun) in C57BL/6 mice increased greatly apoE hepatic mRNA and plasma levels, increased plasma cholesterol, triglyceride, and very low density lipoprotein levels, and resulted in the accumulation of discoidal high density lipoprotein particles. A similar but more severe phenotype was generated by overexpression of the mouse apoE in C57BL/6 mice, suggesting that dyslipidemia induced by dn-c-Jun was the result of apoE overexpression. Unexpectedly, infection of apoE(-/-) mice with adenovirus expressing dn-c-Jun reduced plasma cholesterol by 70%, suggesting that dn-c-Jun affected other genes that control plasma cholesterol levels. To identify these genes, we performed whole genome expression analysis (34,000 genes) of isolated livers from two groups of five apoE(-/-) mice, infected with adenoviruses expressing either the dn-c-Jun or the green fluorescence protein. Bioinformatic analysis and Northern blotting validation revealed that dn-c-Jun increased 40-fold the apoE mRNA and reduced by 70% the Scd-1 (stearoyl-CoA-desaturase 1) mRNA. The involvement of Scd-1 in lowering plasma cholesterol was confirmed by restoration of high cholesterol levels of apoE(-/-) mice following coinfection with adenoviruses expressing dn-c-Jun and Scd-1. In conclusion, dn-c-Jun appears to trigger two opposing events in mice that affect plasma cholesterol and triglyceride levels as follows: one results in apoE overexpression and triggers dyslipidemia and the other results in inhibition of Scd-1 and offsets dyslipidemia.

Rapid turnover of apolipoprotein C-III-containing triglyceride-rich lipoproteins contributing to the formation of LDL subfractions

The atherogenicity theory for triglyceride-rich lipoproteins (TRLs; VLDL + intermediate density lipoprotein) generally cites the action of apolipoprotein C-III (apoC-III), a component of some TRLs, to retard their metabolism in plasma. We studied the kinetics of multiple TRL and LDL subfractions according to the content of apoC-III and apoE in 11 hypertriglyceridemic and normolipidemic persons. The liver secretes mainly two types of apoB lipoproteins: TRL with apoC-III and LDL without apoC-III. Approximately 45% of TRLs with apoC-III are secreted together with apoE. Contrary to expectation, TRLs with apoC-III but not apoE have fast catabolism, losing some or all of their apoC-III and becoming LDL. In contrast, apoE directs TRL flux toward rapid clearance, limiting LDL formation. Direct clearance of TRL with apoC-III is suppressed among particles also containing apoE. TRLs without apoC-III or apoE are a minor, slow-metabolizing precursor of LDL with little direct removal. Increased VLDL apoC-III levels are correlated with increased VLDL production rather than with slow particle turnover. Finally, hypertriglyceridemic subjects have significantly greater production of apoC-III-containing VLDL and global prolongation in residence time of all particle types. ApoE may be the key determinant of the metabolic fate of atherogenic apoC-III-containing TRLs in plasma, channeling them toward removal from the circulation and reducing the formation of LDLs, both those with apoC-III and the main type without apoC-III.

Differential effects of apolipoprotein E isoforms on lipolysis of very low-density lipoprotein triglycerides

Apolipoprotein (apo) E plays a key role in lipoprotein metabolism and has been proposed to modulate triglyceride (TG) lipolysis. However, no systematic investigation on lipolysis using all 3 isoforms of apoE has been performed. To clarify the role of common human apoE isoforms in the lipolysis of very low-density lipoprotein (VLDL) TGs, we overexpressed human apoE isoforms in apoE and low-density lipoprotein receptor–deficient mice using adenoviral-mediated gene transfer and used VLDL particles obtained from these mice for in vitro lipolysis assay. Overexpression of apoE, regardless of its isoforms, increased the TG content of VLDL in mice in vivo. In vitro analysis of the effect of apoE on lipolysis revealed that irrespective of its isoforms, apoE did inhibit TG lipolysis at every concentration of apoE examined, and this inhibitory effect became more pronounced as the apoE content of VLDL increased. No difference was observed in TG lipolysis activity among isoforms at low apoE/TG ratio; however, intermediate ratios of apoE/TG, which reflect physiologic VLDL apoE/TG ratios, demonstrated a significantly greater level of lipolysis inhibition in apoE2, but less so in apoE4 compared with other isoforms. This differential effect by apoE isoforms on lipolysis was attenuated at higher apoE/TG ratios; nevertheless, apoE2 still inhibited lipolysis significantly more than did apoE4. Enrichment of VLDL with apoE decreased both the apoC contents and apoC-II/C-III ratios of VLDL, contributing, at least in part, to the inhibitory function of apoE on lipolysis. The present study clarifies the differential lipolysis-modulating effect of apoE isoforms, which would help explain the difference in pre- and postprandial TG levels among humans carrying different apoE isoforms.

Apolipoprotein E inhibition of vascular hyperplasia and neointima formation requires inducible nitric oxide synthase

Previous studies have shown apolipoprotein E (apoE) recruitment to medial layers of carotid arteries after vascular injury in vivo and apoE activation of inducible nitric oxide synthase (iNOS) in smooth muscle cells in vitro. This investigation explored the relationship between medial apoE recruitment and iNOS activation in protection against neointimal hyperplasia. ApoE was present in both neointimal-resistant C57BL/6 mice and neointimal-susceptible FVB/N mice 24 h after carotid denudation, but iNOS expression was observed only in the neointimal-resistant C57BL/6 mice. However, iNOS was not observed in apoE-defective C57BL/6 mice. In contrast, overexpression of apoE in FVB/N mice activated iNOS expression in the injured vessels, resulting in protection against neointimal hyperplasia. ApoE and iNOS were colocalized in the medial layer of neointimal-resistant mouse strains. Endothelial denudation of carotid arteries in the iNOS-deficient NOS2(-/-) mice did not increase neointimal hyperplasia but significantly increased medial thickness and area. The iNOS-specific inhibitor also abrogated the apoE protective effects on vascular response to injury in apoE-overexpressing FVB/N mice. Thus, injury-induced activation of iNOS requires apoE recruitment. Moreover, both apoE and iNOS are necessary for the suppression of cell proliferation, and apoE recruitment without iNOS expression resulted in medial hyperplasia without cell migration to the intima.

A 13 kDa carboxy‐terminal fragment of ApoE stabilizes Abeta hexamers

The pathological role of ApoE4 in Alzheimer's disease (AD) is not fully elucidated yet but there is strong evidence that ApoE is involved in Abeta deposition, which is an early hallmark of AD neuropathology. Overexpression of ApoE in neuroblastoma cells (Neuro2a) leads to the generation of an intracellular 13 kDa carboxy-terminal fragment of ApoE comparable to fragments seen in brains of AD patients. ApoE4 generates more of this fragment than ApoE2 and E3 suggesting a potential pathological role of these fragments in Alzheimer's disease. Analysis of this intracellular ApoE4 fragment by protease digest followed by MALDI-TOF mass spectrometry showed the proteolytic cleavage site close to residue 187 of ApoE. We have engineered and expressed the corresponding ApoE fragments in vitro. The recombinant 13 kDa carboxy-terminal fragment inhibited fibril formation of Abeta; this contrasts with the full-length ApoE and the corresponding amino-terminal ApoE fragment. Moreover, we show that the 13 kDa carboxy-terminal fragment of ApoE stabilizes the formation of Abeta hexamers. Complexes of Abeta with the 13 kDa carboxy-terminal ApoE fragment show toxicity in PC12 cells comparable to Abeta fibrils. These data suggest that cleavage of ApoE, leading to the generation of this fragment, contributes to the pathogenic effect of ApoE4 in AD.

ApoC-III deficiency prevents hyperlipidemia induced by apoE overexpression

Adenovirus-mediated overexpression of human apolipoprotein E (apoE) induces hyperlipidemia by stimulating the VLDL-triglyceride (TG) production rate and inhibiting the LPL-mediated VLDL-TG hydrolysis rate. Because apoC-III is a strong inhibitor of TG hydrolysis, we questioned whether Apoc3 deficiency might prevent the hyperlipidemia induced by apoE overexpression in vivo. Injection of 2 x 10(9) plaque-forming units of AdAPOE4 caused severe combined hyperlipidemia in Apoe-/- mice [TG from 0.7 +/- 0.2 to 57.2 +/- 6.7 mM; total cholesterol (TC) from 17.4 +/- 3.7 to 29.0 +/- 4.1 mM] that was confined to VLDL/intermediate density lipoprotein-sized lipoproteins. In contrast, Apoc3 deficiency resulted in a gene dose-dependent reduction of the apoE4-associated hyperlipidemia (TG from 57.2 +/- 6.7 mM to 21.2 +/- 18.5 and 1.5 +/- 1.4 mM; TC from 29.0 +/- 4.1 to 16.4 +/- 9.8 and 2.3 +/- 1.8 mM in Apoe-/-, Apoe-/-.Apoc3+/-, and Apoe-/-.Apoc3-/- mice, respectively). In both Apoe-/- mice and Apoe-/-.Apoc3-/- mice, injection of increasing doses of AdAPOE4 resulted in up to a 10-fold increased VLDL-TG production rate. However, Apoc3 deficiency resulted in a significant increase in the uptake of TG-derived fatty acids from VLDL-like emulsion particles by white adipose tissue, indicating enhanced LPL activity. In vitro experiments showed that apoC-III is a more specific inhibitor of LPL activity than is apoE. Thus, Apoc3 deficiency can prevent apoE-induced hyperlipidemia associated with a 10-fold increased hepatic VLDL-TG production rate, most likely by alleviating the apoE-induced inhibition of VLDL-TG hydrolysis.

Apolipoprotein D expression in human brain reactive astrocytes.

Astrocytosis is a hallmark of damage that frequently occurs during aging in human brain. Astrocytes proliferate in elderly subjects, becoming hypertrophic and highly immunoreactive for glial fibrillary acidic protein (GFAP). These cells are one type that actively responds in the repair and reorganization of damage to the neural parenchyma and are a source of several peptides and growth factors. One of these biomolecules is apolipoprotein D (apo D), a member of the lipocalin family implicated in the transport of small hydrophobic molecules. Although the role of apo D is unknown, increments in brain apo D expression have been observed in association with aging and with some types of neuropathology. We have found an overexpression of apo D mRNA in reactive astrocytes by in situ hybridization in combination with immunohistochemistry for apo D in normal aged human brains. The number of double-labeled cells varied according to the cerebral area and the gliosis grade. The possible significance of this increased synthesis of apo D in reactive astrocytes is discussed in relation to the role of apo D in aging and in glial function.

Overexpression of apolipoprotein J in human fibroblasts protects against cytotoxicity and premature senescence induced by ethanol and tert-butylhydroperoxide.

Human diploid fibroblasts (HDFs) exposed to subcytotoxic stresses under H2O2, tert-butylhydroperoxide (t-BHP), and ethanol (EtOH) undergo stress-induced premature senescence (SIPS) characterized by many biomarkers of HDFs replicative senescence. Among these biomarkers are a growth arrest, an increase in the senescence-associated beta-galactosidase activity, a senescent morphology, an overexpression of p21waf-1 and the subsequent inability to phosphorylate pRb, the presence of the common 4977-bp mitochondrial deletion, and an increase in the steady-state level of several senescence-associated genes such as apolipoprotein J (apo J). Apo J has been described as a survival gene against cytotoxic stress. In order to study whether apo J would be protective against cytotoxicity SIPS and replicative senescence in human fibroblasts, a full-length complementary deoxyribonucleic acid of apo J was transfected into WI-38 HDFs and SV40-transformed WI-38 HDFs. The overexpression of apo J resulted in an increased cell survival after t-BHP and EtOH stresses at cytotoxic concentrations. In addition, when WI-38 HDFs were exposed to 5 subcytotoxic stresses with EtOH or t-BHP, in conditions that were previously shown to induce SIPS, a lower induction of 2 biomarkers of SIPS was observed in HDFs overexpressing apo J. No effect of apo J overexpression was observed on the proliferative life span of HDFs, even if apo J overexpression triggered osteonectin (SPARC) overexpression, which was shown to decrease the mitogenic potential of platelet-derived growth factor but not of other common growth-inducing conditions. Apo J senescence-related overexpression is proposed to have antiapoptotic rather than antiproliferative effects.

Effects of coexpression of the LDL receptor and apoE on cholesterol metabolism and atherosclerosis in LDL receptor-deficient mice

The low density lipoprotein receptor (LDLR) plays a major role in regulation of plasma cholesterol levels as a ligand for apolipoprotein B-100 and apolipoprotein E (apoE). Consequently, LDLR-deficient mice fed a Western-type diet develop significant hypercholesterolemia and atherosclerosis. ApoE not only mediates uptake of atherogenic lipoproteins via the LDLR and other cell-surface receptors, but also directly inhibits atherosclerosis. In this study, we examined the hypothesis that coexpression of the LDLR and apoE would have greater effects than either one alone on plasma cholesterol levels and the development of atherosclerosis in LDLR-deficient mice. LDLR-deficient mice fed a Western-type diet for 10 weeks were injected with recombinant adenoviral vectors encoding the genes for human LDLR, human apoE3, both LDLR and apoE3, or lacZ (control). Plasma lipids were analyzed at several time points after vector injection. Six weeks after injection, mice were analyzed for extent of atherosclerosis by two independent methods. As expected, LDLR expression alone induced a significant reduction in plasma cholesterol due to reduced VLDL and LDL cholesterol levels, whereas overexpression of apoE alone did not reduce plasma cholesterol levels. When the LDLR and apoE were coexpressed in this model, the effects on plasma cholesterol levels were no greater than with expression of the LDLR alone. However, coexpression did result in a substantial increase in large apoE-rich HDL particles. In addition, although the combination of cholesterol reduction and apoE expression significantly reduced atherosclerosis, its effects were no greater than with expression of the LDLR or apoE alone. In summary, in this LDLR-deficient mouse model fed a Western-type diet, there was no evidence of an additive effect of expression of the LDLR and apoE on cholesterol reduction or atherosclerosis.—Kawashiri, M-a., Y. Zhang, D. Usher, M. Reilly, E. Puré, and D. J. Rader. Effects of coexpression of the LDL receptor and apoE on cholesterol metabolism and atherosclerosis in LDL receptor-deficient mice.

Low- and high-density lipoprotein metabolism in HepG2 cells expressing various levels of apolipoprotein E.

To determine the importance of hepatic apolipoprotein (apo) E in lipoprotein metabolism, HepG2 cells were transfected with a constitutive expression vector (pRc/CMV) containing either the complete or the first 474 base pairs of the human apoE cDNA inserted in an antisense orientation, for apoE gene inactivation, or the full-length human apoE cDNA inserted in a sense orientation for overexpression of apoE. Stable transformants were obtained that expressed 15, 24, 226, and 287% the apoE level of control HepG2 cells. The metabolism of low-density lipoprotein (LDL) and high-density lipoprotein-3 (HDL(3)), two lipoprotein classes following both holoparticle and cholesteryl esters (CE)-selective uptake pathways, was compared between all these cells. LDL-protein degradation, an indicator of the holoparticle uptake, was greater in low apoE expressing cells than in control or high expressing cells, while HDL(3)-protein degradation paralleled the apoE levels of the cells (r(2) = 0.989). LDL- and HDL(3)-protein association was higher in low apoE expressing cells compared to control cells. In opposition, LDL- and HDL(3)-CE association was not different from control cells in low apoE expressing cells but rose in high apoE expressing cells. In consequence, the CE-selective uptake (CE/protein association ratio) was positively correlated with the level of apoE expression in all cells for both LDL (r(2) = 0.977) and HDL(3) (r(2) = 0.998). We also show that, although in normal and low apoE expressor cells, 92% of LDL- and 80% HDL(3)-CE hydrolysis is sensitive to chloroquine suggesting a pathway linked to lysosomes for both lipoproteins, cells overexpressing apoE lost 60% of chloroquine-sensitive HDL(3)-CE hydrolysis without affecting that of LDL-CE. Thus, the level of apoE expression in HepG2 cells determines the fate of LDL and HDL(3).

Hepatic apolipoprotein E expression promotes very low density lipoprotein-apolipoprotein B production in vivo in mice

In addition to its role in the uptake of apolipoprotein B (apoB)-containing lipoproteins, apoE promotes hepatic very low density lipoprotein-triglyceride (VLDL-TG) production in animal models. However, it is not known if apoE increases the amount of TG per VLDL particle or the number of VLDL particles secreted. VLDL-apoB production is a measure of the rate of VLDL particle secretion. We determined the effects of apoE deficiency and apoE overexpression on VLDL-apoB production in mice. [35S]methionine was injected into endogenously label VLDL-apoB and Triton WR-1339 was simultaneously injected to block the catabolism of VLDL. Compared with wild-type mice, the VLDL-apoB production rate was decreased by 33% in apoE-deficient mice. Conversely, VLDL-apoB production was increased by 48% in mice overexpressing apoE compared with controls. Nascent VLDL, obtained from post-Triton plasma, had a decreased, not increased, content of TG per apoB in the apoE-overexpressing group compared with the control group. This study demonstrates that hepatic apoE expression increases the output of VLDL triglyceride by increasing the production rate of VLDL-apoB, suggesting that hepatic apoE influences the number of VLDL particles secreted by the liver.—Maugeais, C., U. J. F. Tietge, K. Tsukamoto, J. M. Glick, and D. J. Rader. Hepatic apolipoprotein E expression promotes very low density lipoprotein-apolipoprotein B production in vivo in mice. J. Lipid Res. 2000. 41: 1673–1679.

Effect of Overexpression of Human Apo A-I in C57BL/6 and C57BL/6 Apo E–Deficient Mice on 2 Lipoprotein-Associated Enzymes, Platelet-Activating Factor Acetylhydrolase and Paraoxonase

Various mechanisms may contribute to the antiatherogenic potential of apolipoprotein A-I (apo A-I) and high density lipoproteins (HDLs). Therefore, the effect of adenovirus-mediated human apo A-I gene transfer or human apo A-I transgenesis on platelet-activating factor acetylhydrolase (PAF-AH) and arylesterase/paraoxonase (PON1) was studied in C57BL/6 and C57BL/6 apo E(-/-) mice. Human apo A-I transgenesis in C57BL/6 mice resulted in a 4.2-fold (P<0.0001) increase of PAF-AH and a 1.7-fold (P=0.0012) increase of PON1 activity. The apo E deficiency was associated with a 1.6-fold (P=0.008) lower PAF-AH and a 2.0-fold (P=0.012) lower PON1 activity. Human apo A-I transgenesis in C57BL/6 apo E(-/-)mice increased PAF-AH and PON1 activity by 2.1-fold (P=0.01) and 2.5-fold (P=0.029), respectively. After adenovirus-mediated gene transfer of human apo A-I into C57BL/6 apo E(-/-)mice, a strong correlation between human apo A-I plasma levels and PAF-AH activity was observed at day 6 (r=0.92, P<0.0001). However, PON1 activity failed to increase, probably as a result of cytokine-mediated inhibition of PON 1 expression. In conclusion, this study indicates that overexpression of human apo A-I increases HDL-associated PAF-AH activity. PON1 activity was also increased in human apo A-I transgenic mice, but not after human apo A-I gene transfer, a result that was probably related to cytokine production induced in the liver by the adenoviral vectors. Increased levels of these HDL-associated enzymes may contribute to the anti-inflammatory and antioxidative potential of HDL and thereby to the protection conferred by HDL against atherothrombosis.

Hepatic expression of apolipoprotein E inhibits progression of atherosclerosis without reducing cholesterol levels in LDL receptor-deficient mice.

Apolipoprotein E (apoE) is a multifunctional protein synthesized by the liver and by tissue macrophages. Plasma apoE (derived primarily from the liver) regulates plasma lipoprotein metabolism, but macrophage-derived apoE was shown to slow the progression of atherosclerosis independent of plasma lipid levels. We utilized liver-directed gene transfer to test the hypothesis that hepatic expression of human apoE would inhibit atherogenesis even in a model in which apoE expression has little effect on plasma lipoproteins. LDL receptor-deficient mice fed a western-type diet for 5 weeks were injected with a second-generation recombinant adenovirus encoding human apoE3 or control virus. Plasma cholesterol levels were not significantly different in the two groups of mice after virus injection. Four weeks after injection, atherosclerosis was examined using three independent assays. Expression of apoE was associated with significantly reduced atherosclerosis compared with control mice in both the aortic arch (decreased by 43%) and the aortic root (decreased by 59%). In summary, hepatic overexpression of apoE inhibited progression of atherosclerosis in LDL receptor-deficient mice without reducing plasma cholesterol levels. This finding indicates that liver-derived plasma apoE can influence early atherogenesis through mechanisms other than modulation of lipoprotein metabolism and that liver-directed gene transfer and overexpression of apoE may be a therapeutic approach to atherosclerosis.

Overexpression of human hepatic lipase and ApoE in transgenic rabbits attenuates response to dietary cholesterol and alters lipoprotein subclass distributions.

The effect of the expression of human hepatic lipase (HL) or human apoE on plasma lipoproteins in transgenic rabbits in response to dietary cholesterol was compared with the response of nontransgenic control rabbits. Supplementation of a chow diet with 0.3% cholesterol and 3.0% soybean oil for 10 weeks resulted in markedly increased levels of plasma cholesterol and VLDL and IDL in control rabbits as expected. Expression of either HL or apoE reduced plasma cholesterol response by 75% and 60%, respectively. The HL transgenic rabbits had substantial reductions in medium and small VLDL and IDL fractions but not in larger VLDL. LDL levels were also reduced, with a shift from larger, more buoyant to smaller, denser particles. In contrast, apoE transgenic rabbits had a marked reduction in the levels of large VLDLs, with a selective accumulation of IDLs and large buoyant LDLs. Combined expression of apoE and HL led to dramatic reductions of total cholesterol (85% versus controls) and of total VLDL+IDL+LDL (87% versus controls). HDL subclasses were remodeled by the expression of either transgene and accompanied by a decrease in HDL cholesterol compared with controls. HL expression reduced all subclasses except for HDL2b and HDL2a, and expression of apoE reduced large HDL1 and HDL2b. Extreme HDL reductions (92% versus controls) were observed in the combined HL+apoE transgenic rabbits. These results demonstrate that human HL and apoE have complementary and synergistic functions in plasma cholesterol and lipoprotein metabolism.

Overexpression and Accumulation of Apolipoprotein E as a Cause of Hypertriglyceridemia

The molecular mechanisms of hypertriglyceridemia (HTG), a common lipid metabolic disorder in humans, often of genetic origin, are not well understood. In studying the effect of apolipoprotein (apo) E on the metabolism of triglyceride-rich lipoproteins, we found that expressing high plasma levels of human apoE3 in transgenic mice lacking endogenous mouse apoE caused HTG. These transgenic animals had 3-fold higher plasma triglyceride levels, higher very low density lipoproteins (VLDL), and lower high density lipoproteins than did nontransgenics. Removing one or both low density lipoprotein receptor alleles in the apoE3-overexpressing mice caused severe HTG (8-11-fold over nontransgenics) and increased VLDL and decreased low and high density lipoproteins, and apoE3-enriched VLDL were markedly depleted in apoC-II. At least two mechanisms could explain HTG associated with apoE3 overexpression: stimulated VLDL triglyceride production and impaired VLDL lipolysis. The apoE3 mice with HTG had a 50% increase in hepatic VLDL triglyceride production. Furthermore, overexpression of apoE (E2, E3, or E4) in cultured hepatocytes (McA-RH7777 cells) correlated positively with secretion of VLDL into the medium. However, apoE3 overexpression-associated HTG was only partially explained by VLDL overproduction, as lipoprotein lipase-mediated VLDL lipolysis was also decreased 20-86% depending on apoE3 levels, most likely by displacing or masking apoC-II on the particles. In human subjects, HTG correlated positively with increased VLDL triglyceride and plasma and VLDL apoE levels. However, plasma and VLDL apoE correlated negatively with VLDL apoC-II levels and lipoprotein lipase-mediated VLDL lipolysis. Thus, optimal expression of apoE is crucial for normal metabolism of triglyceride-rich lipoproteins, and overexpression and/or accumulation of apoE may contribute to HTG by stimulating VLDL triglyceride production and by impairing VLDL lipolysis. The apoE3-overexpressing mice will be useful for studying the pathophysiology of this disorder.

Conditioned Medium From HepG2 Cells Transfected With Human Apolipoprotein(a) Gene Stimulates Growth of Human Vascular Smooth Muscle Cells

Lipoprotein(a) [Lp(a)] is well known to stimulate growth of vascular smooth muscle cells (VSMCs), resulting in atherosclerosis. Its mechanism is postulated to be decreased in active transforming growth factor (TGF)-beta. However, the exact mechanisms and cellular processing from apolipoprotein(a) [apo(a)] to Lp(a) have not yet been clarified because no cultured cells producing apo(a) are available. Therefore, it is necessary to establish apo(a)-producing cells to study the role of apo(a). We evaluated the effects of overexpression of human apo(a) gene on human aortic VSMC growth. First, we tested whether transfection of apo(a) gene into human hepatoma cells, HepG2 cells, producing human apoB resulted in the formation of Lp(a). Transfection of apo(a) gene into HepG2 cells resulted in detectable levels of Lp(a) in the medium, as assessed by ELISA and Western blot, whereas no Lp(a) was detected in the medium of HepG2 cells transfected with control vector and untransfected HepG2 cells. Expression of apo(a) mRNA was also confirmed by reverse transcription-polymerase chain reaction. In contrast, Western blotting showed a single band detected by specific anti-apo(a) antibody, but not anti-apoB antibody, in the medium of apo(a)-transfected VSMCs. These results demonstrate that Lp(a) can be formed from apo(a) on HepG2 cells, whereas transfection of apo(a) gene into VSMCs resulted in the production of apo(a) alone but not Lp(a). Next, we examined the biological effects of overexpression of apo(a) gene on growth of VSMCs and endothelial cells. Incubation of cultured medium of HepG2 cells transfected with apo(a) gene with human VSMCs or endothelial cells resulted in a significant increase in cell number compared with the conditioned medium of HepG2 cells transfected with control vector. In contrast, transfection of apo(a) gene directly into VSMCs caused no significant effect on VSMC growth. Therefore, we measured TGF-beta concentration in the conditioned medium of VSMCs. However, using ELISA, only latent but not active TGF-beta was detected in the medium of VSMCs. Moreover, addition of neutralizing anti-TGF-beta antibody did not alter VSMC growth. These results suggest that Lp(a) could stimulate growth of VSMCs via the independent mechanisms from the inhibition of TGF-beta activation. Overall, these data demonstrate that overexpression of apo(a) gene in cells producing apoB results in formation of Lp(a), resulting in a mitogenic action on human endothelial cells and VSMCs. These results provide new information to understand the mechanisms of the mitogenic action of Lp(a) and suggest the role of Lp(a) in the pathogenesis of atherosclerosis.

Inhibition of atherosclerosis development in cholesterol-fed human apolipoprotein A-I-transgenic rabbits.

Prospective epidemiological studies support the hypothesis that high levels of high-density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-I limit atherosclerosis development. However, more data from studies with animal models of atherosclerosis that resemble the human disease are required to demonstrate the effect of apo A-I in the inhibition of atherogenesis. The rabbit is a good animal model for human atherosclerosis. Human apo A-I-transgenic rabbits have been produced, and we have evaluated the effect of apo A-I on the development of atherosclerosis in transgenic rabbits fed a cholesterol-rich diet for 14 weeks. Plasma cholesterol levels of atherogenic apo B-containing lipoproteins were similar for transgenic and control rabbits (> 1000 mg/dL), while plasma levels of HDL cholesterol in the transgenic group were always about twice that of the control group (68 +/- 11 versus 37 +/- 3 mg/dL at 14 weeks; P < .001). At the end of the experiment, the amount of aortic surface area covered by lesions as well as the amount of lipid accumulation in the aorta were significantly less in transgenic rabbits compared with the control group (15 +/- 12% versus 30 +/- 8%, P < .0027 for the surface area of the thoracic aorta; 116 +/- 31 versus 247 +/- 39 mumol/g aorta, P < .0068 for cholesterol content in total aorta). Overexpression of human apo A-I in rabbits inhibits the development of atherosclerosis in this animal model that resembles, in many respects, human atherosclerosis.

Transgenic mouse and gene therapy.

In the transgenic mouse, a specific gene can be transduced or deleted to study its function and relation to human diseases. Recently, various lines of transgenic mice that overexpress or lack a specific gene have been established and are available to study the pathophysiology of human diseases, including atherosclerosis, diabetes, and hyperlipidemia. We have established transgenic mouse lines with an integrated rat apolipoprotein (apo) E gene under control of the metallothionein promoter. Overexpression of apoE in the liver reduced plasma cholesterol and triglyceride levels and prevented diet-induced hypercholesterolemia. Another transgenic model with overexpression of apoE under control of the H2 Ld promoter in the arterial wall was established. In this model, the formation of fatty streak lesions was markedly inhibited, suggesting that apoE has antiatherogenic actions. Finally, we discuss gene therapy, which will be an important therapeutic approach to correct genetic abnormalities found in metabolic diseases.

The response-to-retention hypothesis of early atherogenesis.

Many processes have been implicated in early atherogenesis. These include endothelial denudation, injury, or activation, including shear stress–related events; local adherence of platelets; lipoprotein oxidation; lipoprotein aggregation; macrophage chemotaxis and foam cell formation; and smooth muscle cell alterations. Which process, if any, could be regarded as the key event in early atherogenesis, ie, absolutely required, yet also sufficient as the sole pathological stimulus in an otherwise normal artery to provoke a cascade of events leading to lesion formation? The work of many investigators, which we summarize here, strongly supports subendothelial retention of atherogenic lipoproteins as the central pathogenic process in atherogenesis (for prior reviews, see References 1 through 61 2 3 4 5 6 ). Our thesis is that other contributory processes are either not individually necessary or are not sufficient. Most often, they are merely normal, expected responses of otherwise-healthy tissue to the presence of retained lipoproteins. It is instructive to catalog other processes that have been argued to be central to the initiation of atherogenesis. The first is endothelial denudation,7 8 9 injury,10 or activation,11 12 as outlined in the “response-to-injury” hypothesis of Ross, Glomset, and coworkers. Although this important hypothesis has stimulated much of the work that we cite here, there is no definitive evidence in vivo that endothelial injury is either necessary or sufficient for lesion formation. The response-to-injury hypothesis originally presupposed endothelial desquamation as the key event in atherogenesis.7 8 9 It is now clear, however, that developing atheromata are covered by an intact endothelial layer throughout most stages of lesion progression: lipoprotein retention, fatty streak formation, and formation of advanced lesions.5 6 12 13 14 15 In humans, only the most complicated, ulcerated lesions lose their endothelial layer. Furthermore, in some experimental models, an intact endothelium is required …