Endothelium-derived microparticles inhibit angiogenesis in the heart and enhance the inhibitory effects of hypercholesterolemia on angiogenesis

Therapeutic angiogenesis remains unsuccessful in coronary artery disease. It is known that plasma endothelium-derived microparticles (EMPs) are increased in coronary artery disease and that hypercholesterolemia can inhibit angiogenesis. We evaluated the relationship between EMPs and hypercholesterolemia in the impairment of angiogenesis. EMPs isolated from human umbilical vein endothelial cells were injected into low-density lipoprotein receptor-null (LDLr(-/-)) mice fed a Western diet for 2 wk and C57BL6 mice for 6 h or were directly added to the tissue culture media. Hearts isolated from mice were sectioned and cultured, and endothelial tube formation was measured. The expression and phosphorylation of endothelial NO synthase (eNOS) and the generation of NO in the hearts were determined. Angiogenesis was inhibited by pathophysiological concentrations of EMPs but not physiological concentrations of EMPs in hearts from C57BL6 mice. However, angiogenesis was inhibited by EMPs at both physiological and pathophysiological concentrations of EMPs in hearts from hypercholesterolemic LDLr(-/-) mice. Pathophysiological concentrations of EMPs decreased eNOS phosphorylation at Ser(1177) and NO generation without altering eNOS expression in hearts from C57BL6 mice. Both physiological and pathophysiological concentrations of EMPs decreased not only eNOS phosphorylation at Ser(1177) and NO generation, but eNOS expression in hypercholesterolemic hearts from LDLr(-/-) mice. These data demonstrated that pathophysiological concentrations of EMPs could inhibit angiogenesis in hearts by decreasing eNOS activity. EMPs and hypercholesterolemia mutually enhanced their inhibitory effect of angiogenesis by inducing eNOS dysfunction. Our findings suggest a novel mechanism by which hypercholesterolemia impairs angiogenesis.     

Macrophage PLTP is atheroprotective in LDLr-deficient mice with systemic PLTP deficiency

Systemic phospholipid transfer protein (PLTP) is a recognized risk factor for coronary heart disease. In apolipoprotein E-deficient mice, systemic PLTP deficiency is atheroprotective, whereas PLTP overexpression is proatherogenic. As expected, we also observed significantly smaller lesions (P < 0.0001) in hypercholesterolemic double mutant low density lipoprotein receptor-deficient (LDLr(-/-)) PLTP-deficient (PLTP(-/-)) mice compared with LDLr(-/-) mice expressing systemic PLTP. To assess the specific contribution of only macrophage-derived PLTP to atherosclerosis progression, bone marrow transplantation was performed in LDLr(-/-) mice that also lacked systemic PLTP. Groups of double mutant PLTP(-/-)LDLr(-/-) mice were irradiated with 1,000 rad and injected with bone marrow (BM) cells collected from either PLTP(-/-) or wild-type mice. When fed a high-fat diet, BM cell expression of PLTP decreased plasma cholesterol of PLTP(-/-)LDLr(-/-) mice from 878 +/- 220 to 617 +/- 183 mg/dl and increased HDL cholesterol levels from 54 +/- 11 to 117 +/- 19 mg/dl. This decreased total plasma cholesterol and increased HDL cholesterol contributed to the significantly smaller atherosclerotic lesions in both aortas and heart sinus valves observed in these mice. Thus, unlike total systemic PLTP, locally produced macrophage-derived PLTP beneficially alters lipoprotein metabolism and reduces lesion progression in hyperlipidemic mice.     

Role of Toxoplasma gondii HSP70 and Toxoplasma gondii HSP30/bag1 in antibody formation and prophylactic immunity in mice experimentally infected with Toxoplasma gondii.

Production of antibodies against Toxoplasma gondii (T. gondii)-derived stress proteins, T. gondii HSP70 (T.g.HSP70) and T.g.HSP30/bagl, in C57BL/6 and BALB/c mice perorally infected with cysts of the avirulent Fukaya strain of T. gondii was analyzed. Production of anti-T.g.HSP70 IgG antibodies was transient, whereas production of anti-T.g.HSP30/bag1 IgG antibodies persisted after infection in both C57BL/6 and BALB/c mice. C57BL/6 mice, a susceptible strain, predominantly produced IgG antibodies specific for T.g.HSP70, whereas BALB/c mice, a resistant strain, predominantly produced IgG antibodies specific for T.g.HSP30/bag1, after T. gondii infection. Immunization with rT.g.HSP30/bag1 enhanced, whereas immunization with rT.g.HSP70 reduced host protective immunity against T. gondii infection with a cyst-forming avirulent strain, Fukaya, and a virulent strain, RH.     

Immune responses of different mouse strains after challenge with equivalent lethal doses of Toxoplasma gondii

Most immunological studies that utilize different strains of inbred mice following T. gondii infection fail to compensate for differences in host susceptibility to the size of the parasite innoculum. To address this concern, susceptible C57BL/6 and resistant CBA/J mice were orally infected with either an equivalent 50% lethal dose (LD50) of brain cysts of the 76K strain of T. gondii (15 cysts in C57BL/6, 400 cysts in CBA/J) or the same dose of parasites in each mouse strain. C57BL/6 mice receiving 400 cysts (LD50 of CBA/J mice) died post infection, whereas CBA/J mice that received 15 cysts (LD50 of C57BL/6 mice) survived. Parasite loads in the brains and serum Toxoplasma-specific IgG1 titers of LD50-infected C57BL/6 mice were significantly higher than those in LD50- or 15 cysts-infected CBA/J mice, whereas splenocyte proliferation to Toxoplasma antigen and the percentage of CD8 alpha+ T cells were reduced in LD50-infected C57BL/6 mice. In contrast, serum IgG2a and IgM titers, the percentage of gamma delta T cells and IFN-gamma expression of spleen of LD50-infected CBA/J mice were higher than those of either 15 cysts-infected CBA/J mice or LD50-infected C57BL/6 mice. These observations demonstrate that the immune response between LD50-infected C57BL/6 and CBA/J mice was more prominent when compared to C57BL/6 or CBA/J mice receiving the same parasite inoculum. These observations would suggest that caution must be excersized in the planning and interpretation of data when the size of the parasite inoculum has not been adjusted for mouse strain.     

The impaired pregnancy outcome in murine congenital toxoplasmosis is associated with a pro-inflammatory immune response, but not correlated with decidual inducible nitric oxide synthase expression

Congenital toxoplasmosis is associated with adverse pregnancy outcome. Despite the type 1 immune response, C57BL/6 mice are more susceptible than BALB/c mice to Toxoplasma gondii infection. Additionally, successful pregnancy appears to be correlated with type 2 T helper maternal immunity and regulatory T cells. In order to investigate the mechanisms of susceptibility/resistance to congenital toxoplasmosis in mice with different genetic backgrounds and the influence of inducible nitric oxide synthase in pregnancy outcome, groups of C57BL/6, BALB/c and C57BL/6 iNOS(-/-) females were orally infected with T. gondii ME-49 strain on day 1 of pregnancy and were sacrificed on day 8 p.i. and day 19 p.i. The uterus and placenta were evaluated for the foetal resorption rate, parasite load, immunological and histological changes. C57BL/6 mice presented inflammatory foci in the decidua (endometrium) of the uterus at a higher frequency than BALB/c mice on day 8 p.i., and a large number of pregnant C57BL/6 mice presented necrotic implantation sites. The parasite was seldom found in the uterus or placenta of either lineage of mice. Interestingly, there was no observed difference in inducible nitric oxide synthase expression in the uterus and placenta of infected mice. In addition, higher levels of TNF-α were detected in serum samples from C57BL/6 mice compared with BALB/c mice. Accordingly, C57BL/6 mice presented with levels of 90% abortion compared with 50% in BALB/c mice on day 19 p.i. C57BL/6 iNOS(-/-) mice showed low placental parasite counts and high absorption rates, similar to wild type mice. The data suggest that the impaired pregnancy outcome due to T. gondii infection in C57BL/6 mice could be associated with a higher inflammatory response leading to cell apoptosis and necrosis of implantation sites compared with BALB/c mice, and this phenomenon was not due to inducible nitric oxide synthase expression in the decidua.     

Exacerbated susceptibility to infection-stimulated immunopathology in CD1d-deficient mice

Mice lacking functional CD1d genes were used to study mechanisms of resistance to the protozoan parasite Toxoplasma gondii. Wild-type (WT) BALB/c mice, CD1d-deficient BALB/c mice, and WT C57BL/6 mice all survived an acute oral infection with a low dose of mildly virulent strain ME49 T. gondii cysts. In contrast, most CD1d-deficient C57BL/6 mice died within 2 wk of infection. Despite having parasite burdens that were only slightly higher than WT mice, CD1d-deficient C57BL/6 mice displayed greater weight loss and intestinal pathology. In C57BL/6 mice, CD4(+) cells can cause intestinal pathology during T. gondii infection. Compared with WT mice, infected CD1d-deficient C57BL/6 mice had higher frequencies and numbers of activated (CD44(high)) CD4(+) cells in mesenteric lymph nodes. Depletion of CD4(+) cells from CD1d-deficient mice reduced weight loss and prolonged survival, demonstrating a functional role for CD4(+) cells in their increased susceptibility to T. gondii infection. CD1d-deficient mice are deficient in Valpha14(+) T cells, a major population of NKT cells. Involvement of these cells in resistance to T. gondii was investigated using gene-targeted Jalpha18-deficient C57BL/6 mice, which are deficient in Valpha14(+) T cells. These mice did not succumb to acute infection, but experienced greater weight loss and more deaths than B6 mice during chronic infection, indicating that Valpha14(+) cells contribute to resistance to T. gondii. The data identify CD4(+) cells as a significant component of the marked susceptibility to T. gondii infection observed in CD1d-deficient C57BL/6 mice, and establish T. gondii as a valuable tool for deciphering CD1d-dependent protective mechanisms.     

Gene expression profiles in genetically different mice infected with Toxoplasma gondii: ALDH1A2, BEX2, EGR2, CCL3 and PLAU

Toxoplasma gondii can modulate host cell gene expression; however, determining gene expression levels in intermediate hosts after T. gondii infection is not known much. We selected 5 genes (ALDH1A2, BEX2, CCL3, EGR2 and PLAU) and compared the mRNA expression levels in the spleen, liver, lung and small intestine of genetically different mice infected with T. gondii. ALDH1A2 mRNA expressions of both mouse strains were markedly increased at day 1-4 postinfection (PI) and then decreased, and its expressions in the spleen and lung were significantly higher in C57BL/6 mice than those of BALB/c mice. BEX2 and CCR3 mRNA expressions of both mouse strains were significantly increased from day 7 PI and peaked at day 15-30 PI (P<0.05), especially high in the spleen liver or small intestine of C57BL/6 mice. EGR2 and PLAU mRNA expressions of both mouse strains were significantly increased after infection, especially high in the spleen and liver. However, their expression patterns were varied depending on the tissue and mouse strain. Taken together, T. gondii-susceptible C57BL/6 mice expressed higher levels of these 5 genes than did T. gondii-resistant BALB/c mice, particularly in the spleen and liver. And ALDH1A2 and PLAU expressions were increased acutely, whereas BEX2, CCL3 and EGR2 expressions were increased lately. Thus, these demonstrate that host genetic factors exert a strong impact on the expression of these 5 genes and their expression patterns were varied depending on the gene or tissue.     

The induction of acute ileitis by a single microbial antigen of Toxoplasma gondii

The role of specific microbial Ags in the induction of experimental inflammatory bowel disease is poorly understood. Oral infection of susceptible C57BL/6 mice with Toxoplasma gondii results in a lethal ileitis within 7-9 days postinfection. An immunodominant Ag of T. gondii (surface Ag 1 (SAG1)) that induces a robust B and T cell-specific response has been identified and a SAG1-deficient parasite (Deltasag1) engineered. We investigated the ability of Deltasag1 parasite to induce a lethal intestinal inflammatory response in susceptible mice. C57BL/6 mice orally infected with Deltasag1 parasites failed to develop ileitis. In vitro, the mutant parasites replicate in both enterocytes and dendritic cells. In vivo, infection with the mutant parasites was associated with a decrease in the chemokine and cytokine production within several compartments of the gut-associated cell population. RAG-deficient (RAG1(-/-)) mice are resistant to the development of the ileitis after T. gondii infection. Adoptive transfer of Ag-specific CD4(+) effector T lymphocytes isolated from C57BL/6-infected mice into RAG(-/-) mice conferred susceptibility to the development of the intestinal disease. In contrast, CD4(+) effector T lymphocytes from mice infected with the mutant Deltasag1 strain failed to transfer the pathology. In addition, resistant mice (BALB/c) that fail to develop ileitis following oral infection with T. gondii were rendered susceptible following intranasal presensitization with the SAG1 protein. This process was associated with a shift toward a Th1 response. These findings demonstrate that a single Ag (SAG1) of T. gondii can elicit a lethal inflammatory process in this experimental model of pathogen-driven ileitis.     

Deficiency in apolipoprotein E has a protective effect on diet-induced nonalcoholic fatty liver disease in mice

Apolipoprotein E (apoE) mediates the efficient catabolism of the chylomicron remnants very low-density lipoprotein and low-density lipoprotein from the circulation, and the de novo biogenesis of high-density lipoprotein. Lipid-bound apoE is the natural ligand for the low-density lipoprotein receptor (LDLr), LDLr-related protein 1 and other scavenger receptors. Recently, we have established that deficiency in apoE renders mice resistant to diet-induced obesity. In the light of these well-documented properties of apoE, we sought to investigate its role in the development of diet-induced nonalcoholic fatty liver disease (NAFLD). apoE-deficient, LDLr-deficient and control C57BL/6 mice were fed a western-type diet (17.3% protein, 48.5% carbohydrate, 21.2% fat, 0.2% cholesterol, 4.5 kcal·g(-)) for 24 weeks and their sensitivity to NAFLD was assessed by histological and biochemical methods. apoE-deficient mice were less sensitive than control C57BL/6 mice to diet-induced NAFLD. In an attempt to identify the molecular basis for this phenomenon, biochemical and kinetic analyses revealed that apoE-deficient mice displayed a significantly delayed post-prandial triglyceride clearance from their plasma. In contrast with apoE-deficient mice, LDLr-deficient mice fed a western-type diet for 24 weeks developed significant accumulation of hepatic triglycerides and NAFLD, suggesting that apoE-mediated hepatic triglyceride accumulation in mice is independent of LDLr. Our findings suggest a new role of apoE as a key peripheral contributor to hepatic lipid homeostasis and the development of diet-induced NAFLD.     

Effects of iNOS inhibitor on IFN-gamma production and apoptosis of splenocytes in genetically different strains of mice infected with Toxoplasma gondii

To evaluate the role of nitric oxide (NO) in IFN-gamma production and apoptosis of splenocytes in genetically different strains of mice with toxoplasmosis, BALB/c (a toxoplasmosis resistant strain) and C57BL/6 (a toxoplasmosis susceptible strain) mice were infected with Toxoplasma gondii cysts orally and subsequently injected intraperitoneally with aminoguanidine, an iNOS inhibitor (AG; 35 mg/kg per mouse daily for 14 days). When BALB/c or C57BL/6 mice were infected with T. gondii without AG treatment, number of brain cysts, NO and IFN-gamma production by splenocytes, and percentages of apoptotic splenocytes were increased compared to uninfected control mice without AG treatment. AG treatment increased the number of brain cysts, and reduced NO and IFN-gamma production in T. gondii-infected C57BL/6 mice. In contrast, in T. gondii-infected BABL/c mice, the number of brain cysts, and NO and IFN-gamma production of splenocytes was not altered by treatment with AG. However, the percentages of apoptotic splenocytes in T. gondii-infected BALB/c or C57BL/6 mice were not affected by AG treatment. These results suggest that NO modulates IFN-gamma production in T. gondii-infected C57BL/6 mice, and that NO is involved in mediating a protective response in toxoplasmosis susceptible, but not resistant, mice strain during acute infection.     

Inhibition of inducible nitric oxide synthase exacerbates chronic cerebral toxoplasmosis in Toxoplasma gondii-susceptible C57BL/6 mice but does not reactivate the latent disease in T. gondii-resistant BALB/c mice

Infection of C57BL/6 mice with Toxoplasma gondii leads to progressive and ultimately fatal chronic Toxoplasma encephalitis (TE). Genetic deletion or inhibition of inducible nitric oxide synthase (iNOS) from the beginning of infection increased the number of T. gondii cysts in the brain and markedly reduced the time-to-death in this mouse strain. In the present study, we addressed whether iNOS also contributes to the control of intracerebral parasites in a clinically stable latent infection that develops in T. gondii-resistant BALB/c mice after resolution of the acute phase of TE. iNOS was expressed in the inflammatory cerebral infiltrates of latently infected BALB/c mice, but the number of iNOS+ cells was significantly lower than in the brains of chronically infected T. gondii-susceptible C57BL/6 mice. In BALB/c mice with latent TE (> 30 days of infection), treatment with the iNOS inhibitors L-N6-iminoethyl-lysine or L-nitroarginine-methylester for < or = 40 days did not result in an increase of the intracerebral parasitic load and a reactivation of the disease, despite the presence of iNOS-suppressive inhibitor levels in the brain. However, L-nitroarginine-methylester treatment had remarkably toxic effects and induced a severe wasting syndrome with high mortality. In contrast to BALB/c mice, L-N6-iminoethyl-lysine treatment rapidly exacerbated the already established chronic TE of C57BL/6 mice. Thus, the containment of latent toxoplasms in T. gondii-resistant BALB/c mice is independent of iNOS, whereas the temporary control of intracerebral parasites in T. gondii-susceptible C57BL/6 mice with chronic TE requires iNOS activity.     

Apolipoprotein A-I mimetic peptide inhibits atherosclerosis by altering plasma metabolites in hypercholesterolemia

An apolipoprotein A-I mimetic peptide, D-4F, has been shown to improve vasodilation and inhibit atherosclerosis in hypercholesterolemic low-density lipoprotein receptor-null (LDLr(-/-)) mice. To study the metabolic variations of D-4F ininhibiting atherosclerosis, metabonomics, a novel system biological strategy to investigate the pathogenesis, was developed. Female LDLr(-/-) mice were fed a Western diet and injected with or without D-4F intraperitoneally. Atherosclerotic lesion formation was measured, whereas plasma metabolic profiling was obtained on the basis of ultra-high-performance liquid chromatography in tandem with time-of-flight mass spectrometry operating in both positive and negative ion modes. Data were processed by multivariate statistical analysis to graphically demonstrate metabolic changes. The partial least-squares discriminate analysis model was validated with cross-validation and permutation tests to ensure the model's reliability. D-4F significantly inhibited the formation of atherosclerosis in a time-dependent manner. The metabolic profiling was altered dramatically in hypercholesterolemic LDLr(-/-) mice, and a significant metabolic profiling change in response to D-4F treatment was observed in both positive and negative ion modes. Thirty-six significantly changed metabolites were identified as potential biomarkers. A series of phospholipid metabolites, including lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE), phosphatidylcholine (PC), phatidylethanolamine (PE), sphingomyelin (SM), and diacylglycerol (DG), particularly the long-chain LysoPC, was elevated dramatically in hypercholesterolemic LDLr(-/-) mice but reduced by D-4F in a time-dependent manner. Quantitative analysis of LysoPC, LysoPE, PC, and DG using HPLC was chosen to validate the variation of these potential biomarkers, and the results were consistent with the metabonomics findings. Our findings demonstrated that D-4F may inhibit atherosclerosis by regulating phospholipid metabolites specifically by decreasing plasma long-chain LysoPC.     

Interleukin-4 deficiency promotes gallstone formation

Feeding interleukin-4 (IL-4) deficient C57BL/6 LDL receptor (LDLr)(-/-) mice a modified diet to investigate the role of this cytokine in cholesterol metabolism led to an unexpected phenotype. IL-4(-/-) --> LDLr(-/-) mice had enlarged gallbladders and an increased mortality that was preceded by acute body weight loss. To determine if IL-4 deficiency accounted for these findings, C57BL/6 IL-4(+/+) and IL-4(-/-) mice were fed either a normal or modified diet. IL-4 deficiency did not alter bile composition or cause liver toxicity in mice fed a fat-enriched diet. Following 8 weeks of feeding a fat-enriched diet, no gallstones were detected in IL-4(+/+) mice, and only 20% had cholesterol crystals. In contrast, IL-4(-/-) mice had a 100% incidence of gallstones and cholesterol crystals. IL-4(-/-) deficiency also increased serum concentrations of bilirubin following feeding a fat-enriched diet. Therefore, these studies revealed an unexpected finding that IL-4 deficiency predisposes to gallstone formation.     

IL-10 limits parasite burden and protects against fatal myocarditis in a mouse model of Trypanosoma cruzi infection

Chagas' disease is a zoonosis prevalent in Latin America that is caused by the protozoan Trypanosoma cruzi. The immunopathogenesis of cardiomyopathy, the main clinical problem in Chagas' disease, has been extensively studied but is still poorly understood. In this study, we systematically compared clinical, microbiologic, pathologic, immunologic, and molecular parameters in two mouse models with opposite susceptibility to acute myocarditis caused by the myotropic Colombiana strain of T. cruzi: C3H/HeSnJ (100% mortality, uncontrolled parasitism) and C57BL/6J (<10% mortality, controlled parasitism). T. cruzi induced differential polarization of immunoregulatory cytokine mRNA expression in the hearts of C57BL/6J versus C3H/HeSnJ mice; however, most differences were small. The difference in IL-10 expression was exceptional (C57BL/6J 8.7-fold greater than C3H/HeSnJ). Consistent with this, hearts from infected C57BL/6J mice, but not C3H/HeSnJ mice, had a high frequency of total IL-10-producing CD8(+) T cells and both CD4(+) and CD8(+) subsets of IFN-γ(+)IL-10(+) double-producing T cells. Furthermore, T. cruzi infection of IL-10(-/-) C57BL/6J mice phenocopied fatal infection in wild-type C3H/HeSnJ mice with complete loss of parasite control. Adoptive transfer experiments indicated that T cells were a source of protective IL-10. Thus, in this system, IL-10 production by T cells promotes T. cruzi control and protection from fatal acute myocarditis.     

Macrophage-specific expression of group IIA sPLA2 results in accelerated atherogenesis by increasing oxidative stress

Group IIA secretory phospholipase A2 (sPLA2) is an acute-phase protein mediating decreased plasma HDL cholesterol and increased atherosclerosis. This study investigated the impact of macrophage-specific sPLA2 overexpression on lipoprotein metabolism and atherogenesis. Macrophages from sPLA2 transgenic mice have 2.5 times increased rates of LDL oxidation (thiobarbituric acid-reactive substances formation) in vitro (59 +/- 5 vs. 24 +/- 4 nmol malondialdehyde/mg protein; P < 0.001) dependent on functional 12/15-lipoxygenase (12/15-LO). Low density lipoprotein receptor-deficient (LDLR-/-) mice were transplanted with bone marrow from either sPLA2 transgenic mice (sPLA2--> LDLR-/-; n = 19) or wild-type C57BL/6 littermates (C57 BL/6-->LDLR-/-; n = 19) and maintained for 8 weeks on chow and then for 9 weeks on a Western-type diet. Plasma sPLA2 activity and plasma lipoprotein profiles were not significantly different between sPLA2-->LDLR-/- and C57BL/6-->LDLR-/- mice. Aortic root atherosclerosis was increased by 57% in sPLA2-->LDLR-/- mice compared with C57BL/6-->LDLR-/- controls (P < 0.05). Foam cell formation in vitro and in vivo was increased significantly. Urinary, plasma, and aortic levels of the isoprostane 8,12-iso-iPF2alpha-VI and aortic levels of 12/15-LO reaction products were each significantly higher (P < 0.001) in sPLA2-->LDLR-/- compared with C57BL/6-->LDLR-/- mice, indicating significantly increased in vivo oxidative stress in sPLA2--> LDLR-/-. These data demonstrate that macrophage-specific overexpression of human sPLA2 increases atherogenesis by directly modulating foam cell formation and in vivo oxidative stress without any effect on systemic sPLA2 activity and lipoprotein metabolism.     

A novel B-2 suppressor cell regulating susceptibility/resistance of mice to Toxoplasma gondii infection

Irradiation treatment enhanced resistance of C57BL/6, but not BALB/c against Toxoplasma gondii infection. Six Gy-irradiated (IR) C57BL/6 recipients of B-2 cells from T. gondii-infected C57BL/6 died after infection. B-2 suppressor cells from infected C57BL/6 enhanced production of IL-4 and IL-10 in peritoneal exudate cells (PECs), and down-regulated NO release in peritoneal macrophages after infection. On the other hand, B-2 suppressor cells were not detected in a strain, BALB/c, resistant against infection. These data indicated that irradiation-sensitive B-2 cells regulated susceptibility/resistance in mice against T. gondii infection.     

Apolipoprotein E4 in macrophages enhances atherogenesis in a low density lipoprotein receptor-dependent manner

Apolipoprotein E (apoE) and the low density lipoprotein receptor (LDLr) are well recognized determinants of atherosclerosis. In addition to hepatocytes, where both are highly expressed and contribute to plasma lipoprotein clearance, they are expressed in vascular cells and macrophages. In this study, we examined the effects of human apoE isoforms and LDLr levels in atherogenic pathways in primary macrophages ex vivo and atherosclerosis development after bone marrow transfer in vivo using mice expressing human apoE isoforms and different levels of LDLr expression. Increases in LDLr expression significantly increased cholesterol delivery into macrophages in culture, and the effects were more prominent with lipoproteins containing apoE4 than those containing apoE3. Conversely, increased LDLr expression reduced cholesterol efflux in macrophages expressing apoE4 but not in macrophages expressing apoE3. Furthermore, apoE3 protected VLDL from oxidation in vitro more than did apoE4. In LDLr-deficient mice expressing the human apoE4 isoform, Apoe4/4 Ldlr-/-, the replacement of bone marrow cells with those expressing LDLr increased atherosclerotic lesions in a dose-dependent manner compared with mice transplanted with cells having no LDLr. In contrast, atherosclerosis in Apoe3/3 Ldlr-/- mice, expressing the human apoE3 isoform, did not differ by the levels of macrophage LDLr expression. Our results demonstrate that apoE4, but not apoE3, in macrophages enhances atherosclerotic plaque development in mice in an LDLr-dependent manner and suggests that this interaction may contribute to the association of apoE4 with an increased cardiovascular risk in humans.     

Elimination of macrophage-specific apolipoprotein E reduces diet-induced atherosclerosis in C57BL/6J male mice

Apolipoprotein (apo)E is synthesized in atherosclerotic lesions by macrophages, however, its role in lesions is not known. Whereas apoE could exacerbate atherosclerosis by promoting macrophage uptake of cholesterol-rich lipoproteins or modulating protective inflammatory responses, it could also restrict lesion formation by facilitating cholesterol efflux out of lesions. The role of apoE was examined in lethally irradiated male C57BL/6J wild-type (WT) mice that were repopulated with bone marrow cells (BMT) from either identical C57BL/6J mice (WT+WT BMT) or C57BL/6J apoE-deficient mice (WT+E-/- BMT). This enabled us to compare normal mice with mice possessing macrophages that did not express apoE. The participation of macrophage-derived apoE in atherosclerosis was assessed by placing the mice on an atherogenic diet. Male WT+E-/- BMT mice had significantly reduced lesion area in the aortic valves (P < 0.01) compared with male WT+WT BMT mice ( approximately 22,000 vs. approximately 49,000 microm2/section, respectively). Further evaluation revealed that plasma cholesterol, lipoprotein cholesterol distribution, and plasma apoE were similar between the two groups, indicating that these known risk factors did not account for the differences in lesion area. However, the two groups were distinguished by the amount of apoE found in the lesions. ApoE antigen was expressed abundantly in WT+WT BMT lesions, whereas WT+E-/- BMT lesions contained little apoE. These findings indicate that the majority of apoE in lesions is synthesized locally by resident macrophages, and suggest that locally produced apoE can promote diet-induced atherosclerosis in male wild-type mice.     

Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition

The obligate intracellular protozoan Toxoplasma gondii resides within a specialized parasitophorous vacuole (PV), isolated from host vesicular traffic. In this study, the origin of parasite cholesterol was investigated. T. gondii cannot synthesize sterols via the mevalonate pathway. Host cholesterol biosynthesis remains unchanged after infection and a blockade in host de novo sterol biosynthesis does not affect parasite growth. However, simultaneous limitation of exogenous and endogenous sources of cholesterol from the host cell strongly reduces parasite replication and parasite growth is stimulated by exogenously supplied cholesterol. Intracellular parasites acquire host cholesterol that is endocytosed by the low-density lipoprotein (LDL) pathway, a process that is specifically increased in infected cells. Interference with LDL endocytosis, with lysosomal degradation of LDL, or with cholesterol translocation from lysosomes blocks cholesterol delivery to the PV and significantly reduces parasite replication. Similarly, incubation of T. gondii in mutant cells defective in mobilization of cholesterol from lysosomes leads to a decrease of parasite cholesterol content and proliferation. This cholesterol trafficking to the PV is independent of the pathways involving the host Golgi or endoplasmic reticulum. Despite being segregated from the endocytic machinery of the host cell, the T. gondii vacuole actively accumulates LDL-derived cholesterol that has transited through host lysosomes.     

Atherosclerotic lesion formation and triglyceride storage in obese apolipoprotein AI-deficient mice

Obese leptin-deficient (ob/ob) mice have increased levels of high-density lipoprotein (HDL) and a unique lipoprotein referred to as low-density lipoprotein (LDL)/HDL1. When crossed onto an apolipoprotein AI (apoAI)-deficient (-/-) background, ob/ob;apoAI-/- mice accumulate LDL/HDL1 in the absence of traditional HDL. To determine the role of LDL/HDL1 in atherosclerosis, C57BL/6, apoAI-/-, ob/ob and ob/ob;apoAI-/- mice were placed on butterfat diet. After 20 weeks, all four groups had a significant increase in total cholesterol levels. The cholesterol in C57BL/6 mice was carried on very low-density lipoprotein (VLDL) and LDL and, in ob/ob and ob/ob;apoAI-/- mice, on HDL and LDL/HDL1. Atherosclerotic lesion area was similar among C57BL/6, ob/ob and ob/ob;apoAI-/- groups despite their dissimilar lipoprotein profiles. Hepatic triglyceride production and VLDL clearance rates were similar among the four groups. The ob/ob;apoAI-/- group had a significant decrease in liver weight and an increase in white adipose tissue (WAT) weight compared to the ob/ob group. Hepatic scavenger receptor class B type I (SR-BI) levels were decreased in both liver and WAT in ob/ob;apoAI-/- compared to ob/ob mice. Conclusions regarding the atherogenicity of LDL/HDL1 were confounded by the differences in lipoprotein profiles among the four groups. However, our studies provide support for the concept that apoAI and SR-BI assist in the partitioning of lipid from adipose tissue to the liver.