Catherine Desrumaux

Regulated Expression of the Apolipoprotein E/C-I/C-IV/C-II Gene Cluster in Murine and Human Macrophages A CRITICAL ROLE FOR NUCLEAR LIVER X RECEPTORS α AND β

Lipid-loaded macrophage “foam cells” accumulate in the subendothelial space during the development of fatty streaks and atherosclerotic lesions. To better understand the consequences of such lipid loading, murine peritoneal macrophages were isolated and incubated with ligands for two nuclear receptors, liver X receptor (LXR) and retinoic acid receptor (RXR). Analysis of the expressed mRNAs using microarray technology led to the identification of four highly induced genes that encode apolipoproteins E, C-I, C-IV, and C-II. Northern blot analysis confirmed that the mRNA levels of these four genes were induced 2–14-fold in response to natural or synthetic ligands for LXR and/or RXR. The induction of all four mRNAs was greatly attenuated in peritoneal macrophages derived from LXRα/β null mice. The two LXR response elements located within the multienhancers ME.1 and ME.2 were shown to be essential for the induction of apoC-II promoter-reporter genes by ligands for LXR and/or RXR. Finally, immunohistochemical studies demonstrate that apoC-II protein co-localizes with macrophages within murine arterial lesions. Taken together, these studies demonstrate that activated LXR induces the expression of the apoE/C-I/C-IV/C-II gene cluster in both human and murine macrophages. These results suggest an alternative mechanism by which lipids are removed from macrophage foam cells.

Phospholipid Transfer Protein Deficiency Protects Circulating Lipoproteins from Oxidation Due to the Enhanced Accumulation of Vitamin E

Vitamin E is a lipophilic anti-oxidant that can prevent the oxidative damage of atherogenic lipoproteins. However, human trials with vitamin E have been disappointing, perhaps related to ineffective levels of vitamin E in atherogenic apoB-containing lipoproteins. Phospholipid transfer protein (PLTP) promotes vitamin E removal from atherogenic lipoproteins in vitro, and PLTP deficiency has recently been recognized as an anti-atherogenic state. To determine whether PLTP regulates lipoprotein vitamin E contentin vivo, we measured α-tocopherol content and oxidation parameters of lipoproteins from PLTP-deficient mice in wild type, apoE-deficient, low density lipoprotein (LDL) receptor-deficient, or apoB/cholesteryl ester transfer protein transgenic backgrounds. In all four backgrounds, the vitamin E content of very low density lipoprotein (VLDL) and/or LDL was significantly increased in PLTP-deficient mice, compared with controls with normal plasma PLTP activity. Moreover, PLTP deficiency produced a dramatic delay in generation of conjugated dienes in oxidized apoB-containing lipoproteins as well as markedly lower titers of plasma IgG autoantibodies to oxidized LDL. The addition of purified PLTP to deficient plasma lowered the vitamin E content of VLDL plus LDL and normalized the generation of conjugated dienes. The data show that PLTP regulates the bioavailability of vitamin E in atherogenic lipoproteins and suggest a novel strategy for achieving more effective concentrations of anti-oxidants in lipoproteins, independent of dietary supplementation.

Phospholipid transfer protein (PLTP) deficiency reduces brain vitamin E content and increases anxiety in mice

Vitamin E supplementation constitutes a promising strategy in the prevention of neurodegenerative diseases. Here, we show that a phospholipid transfer protein (PLTP) is widely expressed in the brain where it appears to function as a transfer factor for α‐tocopherol, the main isomer of vitamin E. PLTP deficiency results in significant depletion of brain α‐tocopherol in both homozygous (−30.1%, P<0.0002) and heterozygous (−18.0%, P<0.05) PLTP knocked‐out mice. α‐tocopherol depletion in PLTP‐deficient homozygotes is associated with the elevation of lipofuscin (+25% and +450% increases in cortex and substantia nigra, respectively), cholesterol oxides (+54.5%, P<0.05), and cellular peroxides (+32.3%, P<0.01) in the brain. Complete PLTP deficiency in homozygotes is accompanied by increased anxiety as shown by fewer entries (8.3% vs. 44.4% in controls, P<0.01) and less time spent (1.7% vs. 41.3% in controls, P<0.05) in the open arms of an elevated plus‐maze, in the absence of locomotor deterioration. Thus, the vitamin E transfer activity of PLTP appears to be a key process in preventing oxidative damage in the brain, and PLTP‐deficient mice could be a new model of the contribution of oxidative brain injury in the etiology of neurodegenerative diseases.

Mass Concentration of Plasma Phospholipid Transfer Protein in Normolipidemic, Type IIa Hyperlipidemic, Type IIb Hyperlipidemic, and Non–Insulin-Dependent Diabetic Subjects as Measured by a Specific ELISA

Mean plasma phospholipid transfer protein (PLTP) concentrations were measured for the first time by using a competitive enzyme-linked immunosorbent assay. PLTP mass levels and phospholipid transfer activity values, which were significantly correlated among normolipidemic plasma samples (r=0.787, P<0.0001), did not differ between normolipidemic subjects (3.95+/-1.04 mg/L and 575+/-81 nmol. mL-1. h-1, respectively; n=30), type IIa hyperlipidemic patients (4. 06+/-0.84 mg/L and 571+/-43 nmol. mL-1. h-1, respectively; n=36), and type IIb hyperlipidemic patients (3.90+/-0.79 mg/L and 575+/-48 nmol. mL-1. h-1, respectively; n=33). No significant correlations with plasma lipid parameters were observed among the various study groups. In contrast, plasma concentrations of the related cholesteryl ester transfer protein (CETP) were higher in type IIa and type IIb patients than in normolipidemic controls, and significant, positive correlations with total and low density lipoprotein cholesterol levels were noted. Interestingly, plasma PLTP mass concentration and plasma phospholipid transfer activity were significantly higher in patients with non-insulin-dependent diabetes mellitus (n=50) than in normolipidemic controls (6.76+/-1. 93 versus 3.95+/-1.04 mg/L, P<0.0001; and 685+/-75 versus 575+/-81 nmol. mL-1. h-1, P<0.0001, respectively). In contrast, CETP levels did not differ significantly between the 2 groups. Among non-insulin-dependent diabetes mellitus patients, PLTP levels were positively correlated with fasting glycemia and glycohemoglobin levels (r=0.341, P=0.0220; and r=0.382, P=0.0097, respectively) but not with plasma lipid parameters. It is proposed that plasma PLTP mass levels are related to glucose metabolism rather than to lipid metabolism.

Structure and function of the plasma phospholipid transfer protein.

Recent cloning and sequencing of plasma phospholipid transfer protein complementary DNA revealed that phospholipid transfer protein belongs to the lipid transfer/lipopolysaccharide binding protein family that includes the cholesteryl ester transfer protein, the bactericidal permeability increasing protein and the lipopolysaccharide-binding protein. In addition to structural similarities, members of the lipid transfer/lipopolysaccharide-binding protein family might share some common functional properties, and recent studies demonstrated that phospholipid transfer protein can act in several distinct metabolic processes. In particular, the molecular transfer of phospholipids, unesterified cholesterol, alpha-tocopherol and lipopolysaccharides by phospholipid transfer protein suggests that it might be involved both in lipoprotein metabolism and in antimicrobial defence, resulting in a growing interest in this protein.

Atheroprotective Potential of Macrophage-Derived Phospholipid Transfer Protein in Low-Density Lipoprotein Receptor-Deficient Mice Is Overcome by Apolipoprotein AI Overexpression

Objective—Using bone marrow transplantation, we assessed the impact of macrophage-derived phospholipid transfer protein (PLTP) on lesion development in hypercholesterolemic mice that expressed either normal levels of mouse apolipoprotein AI (apoAI) or elevated levels of only human apoAI. Methods and Results—Bone marrow transplantations were performed in low-density lipoprotein receptor–deficient mice (LDLr−/−) that expressed either normal levels of mouse apoAI (msapoAI) or high levels of only human apoAI (msapoAI−/−, LDLr−/−, huapoAITg). Mice were lethally irradiated, reconstituted with either PLTP-expressing or PLTP-deficient bone marrow cells, and fed a high-fat diet over 16 weeks. Macrophage PLTP deficiency increased atherosclerosis in LDLr−/− mice with minimal changes in total plasma cholesterol levels. In contrast, the extent of atherosclerosis in msapoAI−/−, LDLr−/−, huapoAITg mice was not significantly different between groups that had received PLTP−/− or PLTP+/+ bone marrow. In vitro studies indicated that PLTP deficiency led to a significant decrease in α-tocopherol content and increased oxidative stress in bone marrow cells. Conclusions—Our observations suggest an atheroprotective role of macrophage-derived PLTP in mice with normal apoAI plasma levels. The atheroprotective properties of macrophage-derived PLTP were not observable in the presence of elevated plasma concentrations of apoAI.

α-Tocopherol Modulates Phosphatidylserine Externalization in Erythrocytes Relevance in Phospholipid Transfer Protein–Deficient Mice

Objective—The aim of the present study was to assess the effect of α-tocopherol, the main vitamin E isomer on phosphatidylserine (PS) exposure at the surface of circulating erythrocytes, and to determine consequences on erythrocyte properties. Methods and Results—In vitro α-tocopherol enrichment of isolated erythrocytes significantly decreased PS externalization as assessed by lower Annexin V-fluorescein isothiocyanate labeling. Plasma phospholipid transfer protein (PLTP) transfers vitamin E, and both α-and &ggr;-tocopherol accumulated in circulating erythrocytes from PLTP-deficient homozygous (PLTP−/−) mice as compared with wild-type mice. In agreement with in vitro studies, vitamin E–enriched erythrocytes from PLTP−/− mice displayed fewer externalized PS molecules than wild-type controls (−64%, P<0.05). The perturbation of phospholipid membrane asymmetry from PLTP−/− erythrocytes was accompanied by impairment of their procoagulant properties, with a 20% increase in clotting time as compared with wild-type controls (P<0.05). Less pronounced, however still significant, changes were observed in α-tocopherol content, procoagulant properties, and PS externalization in erythrocytes of PLTP-deficient heterozygotes. Finally, whole blood coagulation and circulating level of D-dimer, which reflects increased thrombus formation in vivo, were significantly decreased in PLTP−/− mice compared with wild-type mice. Conclusions—Vitamin E modifies PS externalization in circulating erythrocytes, thus modulating in vivo their PS-dependent procoagulant properties.

Cholesterol accumulation is increased in macrophages of phospholipid transfer protein-deficient mice: normalization by dietary alpha-tocopherol supplementation.

Objective—Phospholipid transfer protein (PLTP) is a multifunctional, extracellular lipid transport protein that plays a major role in lipoprotein metabolism and atherosclerosis. Recent in vivo studies suggested that unlike systemic PLTP, macrophage-derived PLTP would be antiatherogenic. The present study aimed at characterizing the atheroprotective properties of macrophage-derived PLTP. Methods and Results—Peritoneal macrophages were isolated from PLTP-deficient and wild-type mice and their biochemical characteristics were compared. It is shown that macrophages isolated from PLTP-deficient mice have increased basal cholesterol content and accumulate more cholesterol in the presence of LDL compared with wild-type cells. Cholesterol parameters in macrophages of PLTP-deficient mice were normalized by dietary &agr;-tocopherol supplementation. Conclusions—The antiatherogenic properties of macrophage-derived PLTP are related at least in part to its ability to reduce cholesterol accumulation in macrophages through changes in the &agr;-tocopherol content and oxidative status of the cells.

Phospholipid transfer protein deficiency reduces sperm motility and impairs fertility of mouse males

Vitamin E was discovered for its implication in reproductive biology, and its transport in mammalian plasma and brain was shown to be governed by plasma phospholipid transfer protein (PLTP). We show that PLTP deficiency is associated with hypofertility of mouse males but not mouse females, and it accounts for a significant decrease in total number of pups produced over a 2‐month breeding period of PLTP knocked out mice (−32%, P<0.03). PLTP is highly expressed in epididymis of mouse males, and α‐tocopherol, the main vitamin E isomer in vivo, was significantly less abundant in cauda and caput epididymis of PLTP‐deficient mice as compared with wild‐type counterparts (caput: −26%, P<0.05; cauda: −21%, P<0.05). Mature spermatozoa from PLTP‐deficient epididymis were shown to retain an abnormal α‐tocopherol content. PLTP deficiency tended to reduce sperm motility as shown by a 24% reduction in spermatozoa with progressive motility (P<0.02), with no change in other sperm parameters as compared with wild‐type males. Finally, in vitro fertilization rates of wild‐type oocytes with spermatozoa from PLTP‐deficient males were markedly reduced as compared with those measured with spermatozoa from wild‐type males (−60%, P<0.05). It is concluded that PLTP is a new, key factor that determines sperm motility and male fertility.

Development of Abdominal Aortic Aneurysm Is Decreased in Mice with Plasma Phospholipid Transfer Protein Deficiency

Plasma phospholipid transfer protein (PLTP) increases the circulating levels of proatherogenic lipoproteins, accelerates blood coagulation, and modulates inflammation. The role of PLTP in the development of abdominal aortic aneurysm (AAA) was investigated by using either a combination of mechanical and elastase injury at one site of mouse aorta (elastase model) or continuous infusion of angiotensin II in hyperlipidemic ApoE-knockout mice (Ang II model). With the elastase model, complete PLTP deficiency was associated with a significantly lower incidence and a lesser degree of AAA expansion. With the Ang II model, findings were consistent with those in the elastase model, with a lower severity grade in PLTP-deficient mice, an intermediate phenotype in PLTP-deficient heterozygotes, and a blunted effect of the PLTP-deficient trait when restricted to bone marrow-derived immune cells. The protective effect of whole-body PLTP deficiency in AAA was illustrated further by a lesser degree of adventitia expansion, reduced elastin degradation, fewer recruited macrophages, and less smooth muscle cell depletion in PLTP-deficient than in wild-type mice, as evident from comparative microscopic analysis of aorta sections. Finally, cumulative evidence supports the association of PLTP deficiency with reduced expression and activity levels of matrix metalloproteinases, known to degrade elastin and collagen. We conclude that PLTP can play a significant role in the pathophysiology of AAA.