Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by Enhancing Scavenger Receptor Class B1 (SR-BI)-mediated High-density Lipoprotein (HDL)-Cholesteryl Ester Uptake

Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr^−/−, PCPE2^−/− mice, which had elevated HDL levels compared with LDLr^−/− mice with similar LDL concentrations. We found that LDLr^−/−, PCPE2^−/− mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr^−/− mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr^−/−, PCPE2^−/− mice was similar to that reported for LDLr^−/−, apoA-I^−/− mice, which lack any apoA-I/HDL. Furthermore, LDLr^−/−, PCPE2^−/− mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr^−/− mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system.     

Regulation of apoAI processing by procollagen C-proteinase enhancer-2 and bone morphogenetic protein-1

Given the increased prevalence of cardiovascular disease in the world, the search for genetic variations controlling the levels of risk factors associated with the development of the disease continues. Multiple genetic association studies suggest the involvement of procollagen C-proteinase enhancer-2 (PCPE2) in modulating HDL-C levels. Therefore biochemical and mechanistic studies were undertaken to determine whether there might be a basis for a role of PCPE2 in HDL biogenesis. Our studies indicate that PCPE2 accelerates the proteolytic processing of pro-apolipoprotein (apo) AI by enhancing the cleavage of the hexapeptide extension present at the N terminus of apoAI. Surface Plasmon Resonance and immunoprecipitation studies indicate that PCPE2 interacts with BMP-1 and pro-apoAI to form a ternary pro-apoAI/BMP-1/PCPE2 complex. The most favorable interaction among these proteins begins with the association of BMP-1 to pro-apoAI followed by the binding of PCPE2 which further stabilizes the complex. PCPE2 resides, along with apoAI, on the HDL fraction of lipoproteins in human plasma supporting a relationship between HDL and PCPE2. Taken together, the findings from our studies identify a new player in the regulation of apoAI post-translational processing and open a new avenue to the study of mechanisms involved in the regulation of apoAI synthesis, HDL levels, and potentially, cardiovascular disease.     

Adiponectin deficiency suppresses ABCA1 expression and ApoA-I synthesis in the liver

Plasma high density lipoprotein (HDL)-cholesterol levels are inversely correlated with the incidence of cardiovascular diseases. HDL is mainly assembled in the liver through the ATP-binding cassette transporter (ABCA1) pathway. In humans, plasma HDL-cholesterol levels are positively correlated with plasma adiponectin (APN) concentrations. Recently, we reported that APN enhanced apolipoprotein A-I (apoA-I) secretion and ABCA1 expression in HepG2 cells. In the present study, we investigated HDL assembly in APN-knockout (KO) mice. The apoA-I protein levels in plasma and liver were reduced in APN-KO mice compared with wild-type-mice. The ABCA1 expression in liver was also decreased in APN-KO mice. APN deficiency might cause the impaired HDL assembly by decreasing ABCA1 expression and apoA-I synthesis in the liver.     

Overexpression of Human Apolipoprotein A-I Preserves Cognitive Function and Attenuates Neuroinflammation and Cerebral Amyloid Angiopathy in a Mouse Model of Alzheimer Disease

To date there is no effective therapy for Alzheimer disease (AD). High levels of circulating high density lipoprotein (HDL) and its main protein, apolipoprotein A-I (apoA-I), reduce the risk of cardiovascular disease. Clinical studies show that plasma HDL cholesterol and apoA-I levels are low in patients with AD. To investigate if increasing plasma apoA-I/HDL levels ameliorates AD-like memory deficits and amyloid-β (Aβ) deposition, we generated a line of triple transgenic (Tg) mice overexpressing mutant forms of amyloid-β precursor protein (APP) and presenilin 1 (PS1) as well as human apoA-I (AI). Here we show that APP/PS1/AI triple Tg mice have a 2-fold increase of plasma HDL cholesterol levels. When tested in the Morris water maze for spatial orientation abilities, whereas APP/PS1 mice develop age-related learning and memory deficits, APP/PS1/AI mice continue to perform normally during aging. Interestingly, no significant differences were found in the total level and deposition of Aβ in the brains of APP/PS1 and APP/PS1/AI mice, but cerebral amyloid angiopathy was reduced in APP/PS1/AI mice. Also, consistent with the anti-inflammatory properties of apoA-I/HDL, glial activation was reduced in the brain of APP/PS1/AI mice. In addition, Aβ-induced production of proinflammatory chemokines/cytokines was decreased in mouse organotypic hippocampal slice cultures expressing human apoA-I. Therefore, we conclude that overexpression of human apoA-I in the circulation prevents learning and memory deficits in APP/PS1 mice, partly by attenuating neuroinflammation and cerebral amyloid angiopathy. These findings suggest that elevating plasma apoA-I/HDL levels may be an effective approach to preserve cognitive function in patients with AD.     

Phospholipid transfer protein gene knock-out mice have low high density lipoprotein levels, due to hypercatabolism, and accumulate apoA-IV-rich lamellar lipoproteins

Phospholipid transfer protein gene knock-out (Pltp KO) mice have defective transfer of very low density lipoprotein (VLDL) phospholipids into high density lipoprotein (HDL) and markedly decreased HDL levels (Jiang et al. 1999. J. Clin. Invest. 103: 907-914). These animals also accumulated VLDL- and LDL-sized lipoproteins on a high saturated fat diet. The goals of this study were to further characterize the abnormal lipoproteins of Pltp KO mice and to determine the mechanisms responsible for low HDL levels. A lipoprotein fraction enriched in lamellar structures was isolated from the low density lipoprotein (LDL) region and was shown to be phospholipid- and free cholesterol-rich and to have apoA-IV (55%) and apoE (25%) as major apolipoproteins. The lamellar lipoproteins accumulating in these mice probably represent surface material derived from triglyceride-rich lipoproteins (TRL). The HDL was found to be protein-rich (primarily apoA-I) and specifically depleted in phosphatidylcholine (PC) (28% in wild-type mice (WT) vs. 15% in Pltp KO mice, P < 0.001). Unexpectedly, turnover studies using autologous HDL revealed a profound 4-fold increase in the catabolism of HDL protein and cholesteryl ester in Pltp KO mice compared to wild-type, with minor differences in synthesis rates. In contrast, injection of WT mouse HDL into Pltp KO mice showed only a 2-fold increase in fractional catabolism. Reminiscent of the defect in Tangier disease, the failure of transfer of PC from TRL into the HDL fraction results in dramatic hypercatabolism of HDL. These results suggest that defective phospholipid transfer from TRL into HDL, arising from decreased lipolysis or decreased PLTP activity, could lead to hypoalphalipoproteinemia characterized by hypercatabolism of HDL protein. lipoprotein levels, due to hypercatabolism, and accumulate apoA-IV-rich lamellar lipoproteins.     

Apolipoprotein E gene expression is reduced in apolipoprotein A-I transgenic mice

The levels of plasma apolipoprotein (apo) E, an anti-atherogenic protein involved in mammalian cholesterol transport, were found to be 2-3 fold lower in mice over-expressing human apoA-I gene. ApoE is mainly associated with VLDL and HDL-size particles, but in mice the majority of the apoE is associated with the HDL particles. Over-expression of the human apoA-I in mice increases the levels of human apoA-I-rich HDL particles by displacing mouse apoA-I from HDL. This results in lowering of plasma levels of mouse apoA-I. Since plasma levels of apoE also decreased in the apoA-I transgenic mice, the mechanism of apoE lowering was investigated. Although plasma levels of apoE decreased by 2-3 fold, apoB levels remained unchanged. As expected, the plasma levels of human apoA-I were almost 5-fold higher in the apoAI-Tg mice compared to mouse apoA-I in WT mice. If the over-expression of human apoA-I caused displacement of apoE from the HDL, the levels of hepatic apoE mRNA should remain the same in WT and the apoAI-Tg mice. However, the measurements of apoE mRNA in the liver showed 3-fold decreases of apoE mRNA in apoAI-Tg mice as compared to WT mice, suggesting that the decreased apoE mRNA expression, but not the displacement of the apoE from HDL, resulted in the lowering of plasma apoE in apoAI-Tg mice. As expected, the levels of hepatic apoA-I mRNA (transgene) were 5-fold higher in the apoAI-Tg mice. ApoE synthesis measured in hepatocytes also showed lower synthesis of apoE in the apoAI-Tg mice. These studies suggest that the integration of human apoA-I transgene in mouse genome occurred at a site that affected apoE gene expression. Identification of this locus may provide further understanding of the apoE gene expression.     

Persistence of high density lipoprotein particles in obese mice lacking apolipoprotein A-I

Obese mice without leptin (ob/ob) or the leptin receptor (db/db) have increased plasma HDL levels and accumulate a unique lipoprotein referred to as LDL/HDL1. To determine the role of apolipoprotein A-I (apoA-I) in the formation and accumulation of LDL/HDL1, both ob/ob and db/db mice were crossed onto an apoA-I-deficient (apoA-I(-/-)) background. Even though the obese apoA-I(-/-) mice had an expected dramatic decrease in HDL levels, the LDL/HDL1 particle persisted. The cholesterol in this lipoprotein range was associated with both alpha- and beta-migrating particles, confirming the presence of small LDLs and large HDLs. Moreover, in the obese apoA-I(-/-) mice, LDL particles were smaller and HDLs were more negatively charged and enriched in apoE compared with controls. This LDL/HDL1 particle was rapidly remodeled to the size of normal HDL after injection into C57BL/6 mice, but it was not catabolized in obese apoA-I(-/-) mice even though plasma hepatic lipase (HL) activity was increased significantly. The finding of decreased hepatic scavenger receptor class B type I (SR-BI) protein levels may explain the persistence of LDL/HDL1 in obese apoA-I(-/-) mice. Our studies suggest that the maturation and removal of large HDLs depends on the integrity of a functional axis of apoA-I, HL, and SR-BI. Moreover, the presence of large HDLs without apoA-I provides evidence for an apoA-I-independent pathway of cholesterol efflux, possibly sustained by apoE.     

Alteration of negatively charged residues in the 89 to 99 domain of apoA-I affects lipid homeostasis and maturation of HDL

In this study, we investigated the role of positively and negatively charged amino acids within the 89-99 region of apolipoprotein A-I (apoA-I), which are highly conserved in mammals, on plasma lipid homeostasis and the biogenesis of HDL. We previously showed that deletion of the 89-99 region of apoA-I increased plasma cholesterol and phospholipids, but it did not affect plasma triglycerides. Functional studies using adenovirus-mediated gene transfer of two apoA-I mutants in apoA-I-deficient mice showed that apoA-I[D89A/E91A/E92A] increased plasma cholesterol and caused severe hypertriglyceridemia. HDL levels were reduced, and approximately 40% of the apoA-I was distributed in VLDL/IDL. The HDL consisted of mostly spherical and a few discoidal particles and contained preβ1 and α4-HDL subpopulations. The lipid, lipoprotein, and HDL profiles generated by the apoA-I[K94A/K96A] mutant were similar to those of wild-type (WT) apoA-I. Coexpression of apoA-I[D89A/E91A/E92A] and human lipoprotein lipase abolished hypertriglyceridemia, restored in part the α1,2,3,4 HDL subpopulations, and redistributed apoA-I in the HDL2/HDL3 regions, but it did not prevent the formation of discoidal HDL particles. Physicochemical studies showed that the apoA-I[D89A/E91A/E92A] mutant had reduced α-helical content and effective enthalpy of thermal denaturation, increased exposure of hydrophobic surfaces, and increased affinity for triglyceride-rich emulsions. We conclude that residues D89, E91, and E92 of apoA-I are important for plasma cholesterol and triglyceride homeostasis as well as for the maturation of HDL.     

Endothelial lipase is less effective at influencing HDL metabolism in vivo in mice expressing apoA-II

Endothelial lipase (EL) plays an important physiological role in modulating HDL metabolism. Data suggest that plasma contains an inhibitor of EL, and previous studies have suggested that apolipoprotein A-II (apoA-II) inhibits the activity of several enzymes involved in HDL metabolism. Therefore, we hypothesized that apoA-II may reduce the ability of EL to influence HDL metabolism. To test this hypothesis, we determined the effect of EL expression on plasma phospholipase activity and HDL metabolism in human apoA-I and human apoA-I/A-II transgenic mice. Expression of EL in vivo resulted in lower plasma phospholipase activity and significantly less reduction of HDL-cholesterol, phospholipid, and apoA-I levels in apoA-I/A-II double transgenic mice compared with apoA-I single transgenic mice. We conclude that the presence of apoA-II on HDL particles inhibits the ability of EL to influence the metabolism of HDL in vivo.