Low-dose radiation exposure and protection against atherosclerosis in ApoE(-/-) mice: the influence of P53 heterozygosity.

We recently described the effects of low-dose γ-radiation exposures on atherosclerosis in genetically susceptible (ApoE(-/-)) mice with normal p53 function. Doses as low as 25 mGy, given at either early or late stage disease, generally protected against atherosclerosis in a manner distinctly nonlinear with dose. We now report the influence of low doses (25-500 mGy) on atherosclerosis in ApoE(-/-) mice with reduced p53 function (Trp53(+/-)). Single exposures were given at either low or high dose rate (1 or 150 mGy/min) to female C57BL/6J ApoE(-/-) Trp53(+/-) mice. Mice were exposed at either early stage disease (2 months of age) and examined 3 or 6 months later, or at late stage disease (7 months of age) and examined 2 or 4 months later. In unirradiated mice, reduced p53 functionality elevated serum cholesterol and accelerated both aortic root lesion growth and severity in young mice. Radiation exposure to doses as low as 25 mGy at early stage disease, at either the high or the low dose rate, inhibited lesion growth, decreased lesion frequency and slowed the progression of lesion severity in the aortic root. In contrast, exposure at late stage disease produced generally detrimental effects. Both low-and high-dose-rate exposures accelerated lesion growth and high dose rate exposures also increased serum cholesterol levels. These results show that at early stage disease, reduced p53 function does not influence the protective effects against atherosclerosis of low doses given at low dose rate. In contrast, when exposed to the same doses at late stage disease, reduced p53 function produced detrimental effects, rather than the protective effects seen in Trp53 normal mice. As in the Trp53 normal mice, all effects were highly nonlinear with dose. These results indicate that variations in p53 functionality can dramatically alter the outcome of a low-dose exposure, and that the assumption of a linear response with dose for human populations is probably unwarranted.

Injected matrix stimulates myogenesis and regeneration of mouse skeletal muscle after ischaemic injury.

Biomaterial-guided regeneration represents a novel approach for the treatment of myopathies. Revascularisation and the intramuscular extracellular matrix are important factors in stimulating myogenesis and regenerating muscle damaged by ischaemia. In this study, we used an injectable collagen matrix, enhanced with sialyl LewisX (sLeX), to guide skeletal muscle differentiation and regeneration. The elastic properties of collagen and sLeX-collagen matrices were similar to those of skeletal muscle, and culture of pluripotent mESCs on the matrices promoted their differentiation into myocyte-like cells expressing Pax3, MHC3, myogenin and Myf5. The regenerative properties of matrices were evaluated in ischaemic mouse hind-limbs. Treatment with the sLeX-matrix augmented the production of myogenic-mediated factors insulin-like growth factor (IGF)-1, and IGF binding protein-2 and -5 after 3 days. This was followed by muscle regeneration, including a greater number of regenerating myofibres and increased transcription of Six1, M-cadherin, myogenin and Myf5 after 10 days. Simultaneously, the sLeX-matrix promoted increased mobilisation and engraftment of bone marrow-derived progenitor cells, the development of larger arterioles and the restoration of tissue perfusion. Both matrix treatments tended to reduce maximal forces of ischaemic solei muscles, but sLeX-matrix lessened this loss of force and also prevented muscle fatigue. Only sLeX-matrix treatment improved mobility of mice on a treadmill. Together, these results suggest a novel approach for regenerative myogenesis, whereby treatment only with a matrix, which possesses an inherent ability to guide myogenic differentiation of pluripotent stem cells, can enhance the endogenous vascular and myogenic regeneration of skeletal muscle, thus holding promise for future clinical use.

Low-dose radiation exposure and atherosclerosis in ApoE⁻/⁻ mice.

The hypothesis that single low-dose exposures (0.025-0.5 Gy) to low-LET radiation given at either high (about 150 mGy/min) or low (1 mGy/min) dose rate would promote aortic atherosclerosis was tested in female C57BL/6J mice genetically predisposed to this disease (ApoE⁻/⁻). Mice were exposed either at an early stage of disease (2 months of age) and examined 3 or 6 months later or at a late stage of disease (8 months of age) and examined 2 or 4 months later. Changes in aortic lesion frequency, size and severity as well as total serum cholesterol levels and the uptake of lesion lipids by lesion-associated macrophages were assessed. Statistically significant changes in each of these measures were observed, depending on dose, dose rate and disease stage. In all cases, the results were distinctly non-linear with dose, with maximum effects tending to occur at 25 or 50 mGy. In general, low doses given at low dose rate during either early- or late-stage disease were protective, slowing the progression of the disease by one or more of these measures. Most effects appeared and persisted for months after the single exposures, but some were ultimately transitory. In contrast to exposure at low dose rate, high-dose-rate exposure during early-stage disease produced both protective and detrimental effects, suggesting that low doses may influence this disease by more than one mechanism and that dose rate is an important parameter. These results contrast with the known, generally detrimental effects of high doses on the progression of this disease in the same mice and in humans, suggesting that a linear extrapolation of the known increased risk from high doses to low doses is not appropriate.

Macrophage-specific expression of class A scavenger receptors enhances granuloma formation in the absence of increased lipid deposition.

Class A scavenger receptors (SR-A) have several proposed functions that could impact atherosclerosis and inflammatory processes. To define the function of SR-A in vivo, we created C57BL/6 transgenic mice that expressed bovine SR-A under the control of the restricted macrophage promoter, lysozyme (lyso-bSR-A). bSR-A mRNA was present in cultured peritoneal macrophages of transgenic mice and tissues that contain significant macrophages including spleen, lung, and ileum. Functional overexpression of SR-A was demonstrated in peritoneal macrophages both by augmented cholesterol ester deposition in response to AcLDL and enhanced adhesion in transgenic mice compared with nontransgenic littermates. To determine whether macrophage-specific expression of bSR-A regulated inflammatory responses, granulomas were generated by subcutaneous injection of carrageenan. Granuloma size was significantly increased in lyso-bSR-A transgenic mice compared with wild-type littermates [421 +/- 51 mg (n = 11) vs. 127 +/- 22 mg (n = 10), P < 0.001]. However, the larger granulomas in lyso-bSR-A transgenic mice were only associated with an increase in unesterified cholesterol, and not cholesterol esters. Furthermore, granulomas from transgenic mice had an increase in the number of macrophages within the tissue.Therefore, macrophage expression of bSR-A increased presence of this cell type in granulomas without enhancing the deposition of cholesterol esters, consistent with a role of the adhesive property of the protein.

Exogenous interferon-gamma enhances atherosclerosis in apolipoprotein E-/- mice.

A role for interferon-gamma (IFN-gamma) has been implied in the atherogenic process. To determine whether exogenously administered IFN-gamma exerts an effect on the development of atherosclerosis, we intraperitoneally administered either recombinant IFN-gamma (100 U/g body weight) or phosphate buffered saline daily for 30 days to atherosclerosis-susceptible apolipoprotein E-/- mice (16-week-old male mice, n = 11 per group) fed a normal diet. Atherosclerotic lesion size was quantified in the ascending aorta. The number of T lymphocytes and major histocompatibility complex (MHC) class II-positive cells within lesions were also quantified in this region. IFN-gamma administration reduced serum cholesterol concentrations by 15% (P = 0.02). For both groups, the majority of cholesterol was present in very low density lipoproteins, which were modestly reduced in mice receiving IFN-gamma. Despite the decrease in serum cholesterol concentrations, IFN-gamma injections significantly increased lesion size twofold compared to controls (119,980 +/- 18, 536 vs. 59,396 +/- 20,017 micrometer(2); P = 0.038). IFN-gamma also significantly increased the mean number of T lymphocytes (19 +/- 4 vs. 7 +/- 1 cells; P = 0.03) and MHC class II-positive cells (10 +/- 3 vs. 3 +/- 1 cells; P = 0.04) within lesions. These data lend further support to a pro-atherogenic role of IFN-gamma.

Polymorphism of class A scavenger receptors in C57BL/6 mice.

Scavenger receptors class A (SR-A) have been hypothesized to regulate the development of atherosclerotic lesions through recognition of modified low density lipoprotein (LDL) and macrophage adhesion to substrata. Supporting data have been collected from studies using the monoclonal antibody 2F8, an antibody developed from the BALB/c strain-derived macrophage cell line, RAW.264. Although 2F8 immunostained both cultured peritoneal macrophages (MPM) and thymic macrophages from Swiss, BALB/c, and DBA/2 mice, no immunostaining was detected in cells and tissues from C57BL/6 mice, one of the most commonly used atherosclerosis-susceptible mouse strains. Similarly, 2F8 detected SR-A protein in MPM by Western blotting in all strains except C57BL/6. However, a guinea pig antiserum developed to a fusion protein of the extracellular SR-A domain detected appropriately sized bands in all strains. Incubation with 2F8 antagonized acetylated low-density lipoprotein (AcLDL)-induced cholesterol esterification in MPM from BALB/c, Swiss, and DBA/2 strains but had no effect on MPM from C57BL/6 mice. Sequencing of SR-A cDNA from C57BL/6 mice demonstrated complete identity with published sequence in the collagen-like domain. However, four single-residue substitutions were noted in the alpha-helical coiled-coil domain. Site-directed mutagenesis demonstrated that a single substitution (L168S) in this domain accounted for the loss of 2F8 immunoreactivity. Differing reactivities toward a commonly used monoclonal antibody were used to identify polymorphism of SR-A in C57BL/6 mice.

Macrophage colony-stimulating factor rapidly enhances beta-migrating very low density lipoprotein metabolism in macrophages through activation of a Gi/o protein signaling pathway.

Previous studies have examined lipoprotein metabolism by macrophages following prolonged exposure (>24 h) to macrophage colony-stimulating factor (M-CSF). Because M-CSF activates several signaling pathways that could rapidly affect lipoprotein metabolism, we examined whether acute exposure of macrophages to M-CSF alters the metabolism of either native or modified lipoproteins. Acute incubation of cultured J774 macrophages and resident mouse peritoneal macrophages with M-CSF markedly enhanced low density lipoproteins (LDL) and beta-migrating very low density lipoproteins (beta-VLDL) stimulated cholesteryl [(3)H]oleate deposition. In parallel, M-CSF treatment increased the association and degradation of (125)I-labeled LDL or beta-VLDL without altering the amount of lipoprotein bound to the cell surface. The increase in LDL and beta-VLDL metabolism did not reflect a generalized effect on lipoprotein endocytosis and metabolism because M-CSF did not alter cholesterol deposition during incubation with acetylated LDL. Moreover, M-CSF did not augment beta-VLDL cholesterol deposition in macrophages from LDL receptor (-/-) mice, indicating that the effect of M-CSF was mediated by the LDL receptor. Incubation of macrophages with pertussis toxin, a specific inhibitor of G(i/o) protein signaling, had no effect on cholesterol deposition during incubation with beta-VLDL alone, but completely blocked the augmented response promoted by M-CSF. In addition, incubation of macrophages with the direct G(i/o) protein activator, mastoparan, mimicked the effect of M-CSF by enhancing cholesterol deposition in cells incubated with beta-VLDL, but not acetylated LDL. In summary, M-CSF rapidly enhances LDL receptor-mediated metabolism of native lipoproteins by macrophages through activation of a G(i/o) protein signaling pathway. Together, these findings describe a novel pathway for regulating lipoprotein metabolism.

Regulation of acetylated low density lipoprotein uptake in macrophages by pertussis toxin-sensitive G proteins.

Class A scavenger receptors (SR-A) mediate the uptake of modified low density lipoprotein (LDL) by macrophages. Although not typically associated with the activation of intracellular signaling cascades, results with peritoneal macrophages indicate that the SR-A ligand acetylated LDL (AcLDL) promotes activation of cytosolic kinases and phospholipases. These signaling responses were blocked by the treatment of cells with pertussis toxin (PTX) indicating that SR-A activates G(i/o)-linked signaling pathways. The functional significance of SR-A-mediated G(i/o) activation is not clear. In this study, we investigated the potential role of G(i/o) activation in regulating SR-A-mediated lipoprotein uptake. Treatment of mouse peritoneal macrophages with PTX decreased association of fluorescently labeled AcLDL with cells. This inhibition was dependent on the catalytic activity of the toxin confirming that the decrease in AcLDL uptake involved inhibiting G(i/o) activation. In contrast to the inhibitory effect on AcLDL uptake, PTX treatment did not alter beta-VLDL-induced cholesterol esterification or deposition of cholesterol. The ability of polyinosine to completely inhibit AcLDL uptake, and the lack of PTX effect on beta-VLDL uptake, demonstrated that the inhibitory effect is specific for SR-A and not the result of non-specific effects on lipoprotein metabolism. Despite having an effect on an SR-A-mediated lipoprotein uptake, there was no change in the relative abundance of SR-A protein after PTX treatment. These results demonstrate that activation of a PTX-sensitive G protein is involved in a feedback process that positively regulates SR-A function.

Uptake of type IV hypertriglyceridemic VLDL by cultured macrophages is enhanced by interferon-gamma.

Hypertriglyceridemic (HTG) very low density lipoproteins (VLDL) from subjects with type IV hyperlipoproteinemia induce both cholesteryl ester (CE) and triglyceride (TG) accumulation in cultured J774 macrophages. We examined whether the cytokine interferon-gamma (IFN-gamma), which is expressed by lymphocytes in atherosclerotic lesions, would modulate macrophage uptake of HTG -VLDL. Incubation of cells with HTG -VLDL alone significantly increased cellular CE and TG mass 17- and 4.3-fold, respectively, while cellular free cholesterol (FC) was unaffected. Pre-incubation of cells with IFN-gamma (50 U/ml) prior to incubation with HTG -VLDL caused a marked enhancement in cellular CE and TG 27- and 6-fold over no additions (controls), respectively, and a 1.5-fold increase in FC. IFN-gamma increased low density lipoprotein (LDL)-induced cellular CE 2-fold compared to LDL alone. IFN-gamma did not enhance the uptake of type III (apoE2/E2) HTG -VLDL or VLDL from apoE knock-out mice. Incubations in the presence of a lipoprotein lipase (LPL) inhibitor or an acylCoA:cholesterol acyltransferase (ACAT) inhibitor demonstrated that the IFN-gamma-enhanced HTG -VLDL uptake was dependent on LPL and ACAT activities. IFN-gamma significantly increased the binding and degradation of 125I-labeled LDL. Binding studies with 125I-labeled alpha2-macroglobulin, a known LDL receptor-related protein (LRP) ligand, and experiments with copper-oxidized LDL indicated that the IFN-gamma-enhanced uptake was not due to increased expression of the LRP or scavenger receptors. Thus, IFN-gamma may promote foam cell formation by accelerating macrophage uptake of native lipoproteins. IFN-gamma-stimulated CE accumulation in the presence of HTG -VLDL occurs via a process that requires receptor binding-competent apoE and active LPL. IFN-gamma-enhanced uptake of both HTG -VLDL and LDL is mediated by the LDL-receptor and requires ACAT-mediated cholesterol esterification.

Modification of type III VLDL, their remnants, and VLDL from ApoE-knockout mice by p-hydroxyphenylacetaldehyde, a product of myeloperoxidase activity, causes marked cholesteryl ester accumulation in macrophages.

Very low density lipoproteins (VLDLs) from apolipoprotein (apo) E2/E2 subjects with type III hyperlipoproteinemia, VLDL remnants, and VLDL from apoE-knockout (EKO) mice are taken up poorly by macrophages. The present study examined whether VLDL modification by the reactive aldehyde p-hydroxyphenylacetaldehyde (pHA) enhances cholesteryl ester (CE) accumulation by J774A.1 macrophages. pHA is the major product derived from the oxidation of L-tyrosine by myeloperoxidase and is a component of human atherosclerotic lesions. Incubation of J774A.1 cells with native type III VLDL, their remnants, and EKO-VLDL increased cellular CE by only 3-, 5-, and 5-fold, respectively, compared with controls. In striking contrast, cells exposed to VLDL modified by purified pHA (pHA-VLDL) exhibited marked increases in cellular CE of 38-, 47-, and 35-fold, respectively (P95%, CE accumulation induced by copper-oxidized VLDL. These results demonstrate a novel mechanism for the conversion of type III VLDLs, their remnants, and EKO-VLDL into atherogenic particles and suggest that macrophage uptake of pHA-VLDL (1) requires catalytically active lipoprotein lipase, (2) involves acyl coenzyme A:cholesterol acyltransferase-mediated cholesterol esterification, and (3) involves pathways distinct from the SR-A.

Oxidized type IV hypertriglyceridemic VLDL-remnants cause greater macrophage cholesteryl ester accumulation than oxidized LDL.

We have previously shown that very low density lipoproteins (VLDL, Sf 60-400) from subjects with type IV hyperlipoproteinemia (HTG-VLDL) will induce appreciable cholesteryl ester accumulation in cultured macrophages (J774A.1). The present study examined whether copper-mediated oxidative modification of HTG-VLDL and their remnants would further enhance cholesteryl ester accumulation in J774A.1 cells. Incubation with oxidized VLDL-remnants caused the greatest increase in cellular cholesteryl ester concentrations (54-fold) relative to control cells (P = 0.001). HTG-VLDL and VLDL-remnants each induced similar increases in cholesteryl ester levels (32.3- and 35.8-fold, respectively; both P = 0.001), whereas incubation with oxidized HTG-VLDL brought about only a 20.6-fold increase in cholesteryl ester concentrations (P = 0.014). The increase in cellular cholesteryl ester concentrations induced by oxidized VLDL-remnants was significantly higher (P < or = 0.04) than that induced by all other lipoproteins tested including low density lipoprotein (LDL) and oxidized LDL which caused a 6.7- and a 35.1-fold increase (P < or = 0.0002 for both), respectively. Unlike HTG-VLDL and to a lesser extent VLDL-remnants, uptake of oxidized VLDL and oxidized VLDL-remnants did not require catalytically active, cell secreted lipoprotein lipase. Co-incubation with polyinosine, which blocks binding to the type I scavenger receptor, completely inhibited the cholesteryl ester accumulation induced by oxidized HTG-VLDL, oxidized VLDL-remnants and oxidized LDL (P < or = 0.02). We conclude that oxidation of VLDL-remnants significantly enhances macrophage cholesteryl ester accumulation compared to either HTG-VLDL, VLDL-remnants, or oxidized LDL. Uptake of oxidized VLDL and oxidized VLDL-remnants does not require catalytically active lipoprotein lipase, and involves a receptor that can be competed for by polyinosine.

Uptake of type III hypertriglyceridemic VLDL by macrophages is enhanced by oxidation, especially after remnant formation.

We previously showed that hypertriglyceridemic VLDL (HTG-VLDL, Sf 60 to 400) from subjects with type III (E2/E2) hyperlipoproteinemia do not induce appreciable cholesteryl ester (CE) accumulation in cultured macrophages (J774A.1). In the present study, we examined whether oxidation of type III HTG-VLDL would enhance their uptake by J774A.1 cells. Type III HTG-VLDL were oxidized as measured by both conjugated-diene formation and increased electrophoretic mobility on agarose gels. Both LDL and type III HTG-VLDL undergo oxidation, albeit under different kinetic parameters. From the conjugated-diene curve, type III HTG-VLDL, compared with LDL, were found to have a 6-fold longer lag time, to take 6-fold longer to reach maximal diene production, and to produce a 2-fold greater amount of dienes but at half the rate (all P < .005). Incubation of macrophages with either native type III HTG-VLDL or LDL (50 micrograms lipoprotein cholesterol/mL media for 16 hours) caused small increases (4-fold and 2.7-fold, respectively) in cellular CE levels relative to control cells (both P = .0001). After 24 hours of CuSO4 exposure, we found that oxidized type III HTG-VLDL and LDL caused a 9.4-fold and 10.5-fold increase, respectively, in cellular CE levels (P = .0001). We next examined whether extending the exposure period for type III HTG-VLDL to CuSO4 beyond 24 hours would further enhance its ability to induce macrophage CE accumulation. After 48 hours of CuSO4 exposure, type III HTG-VLDL and LDL caused 21.3-fold and 11.6-fold increases, respectively, in cellular CE levels (P = .0001). The cellular CE loading achieved with 48 hour-oxidized type III HTG-VLDL was significantly higher than either 24 hour-oxidized type III HTG-VLDL (2.3-fold, P = .003) or 48 hour-oxidized LDL (1.8-fold, P = .012). There was no significant difference between the CE loading achieved by incubation of cells with either 24 hour-oxidized type III HTG-VLDL, 24 hour-oxidized LDL, or 48 hour-oxidized LDL (P > or = .518). In this study, we also examined whether partial lipolysis (19% to 50% triglyceride hydrolysis) of type III HTG-VLDL to produce remnants would increase the susceptibility of the lipoprotein to oxidative modification and subsequent cellular CE loading. Forty-eight hour-oxidized type III VLDL-remnants stimulated CE accumulation 30.4-fold over baseline (P = .0001). In contrast, nonoxidized type III VLDL-remnants caused the same very low level of CE loading as did native type III HTG-VLDL (P = .680). The increase in cellular CE levels achieved with 48 hour-oxidized type III VLDL-remnants was significantly higher than that achieved with 48 hour-oxidized type III HTG-VLDL (P = .047). In conclusion, we have shown that oxidized type III HTG-VLDL will induce macrophage CE accumulation well above levels achieved with oxidized LDL. In addition, we also showed that by forming a VLDL-remnant before oxidative modification, we can further enhance macrophage CE accumulation. These results provide a potential mechanism for the atherogenicity of type III HTG-VLDL and their remnants.

Probucol, but not MaxEPA fish oil, inhibits mononuclear cell adhesion to the aortic intima in the rat model of atherosclerosis.

We have examined the influence of both dietary fish oil and probucol on monocyte adhesion to the aortic endothelium rats fed an atherogenic diet for 2 weeks. All rats were fed a low-fat diet supplemented with 4% cholesterol, 1% cholic acid, and 0.5% 2-thiouracil. In addition to the atherogenic diet, group 1 (FO; n = 20) received a dietary supplement of the fish oil concentrate MaxEPA (5% w/w); group 2 (CO; n = 20) received a supplement of a control oil with same polyunsaturated-monounsaturated-saturated fatty acid ratio as Max-EPA; and group 3 (PR; n = 20) received both the control oil supplement (5% w/w) and a 1% (w/w) supplement of probucol. Analysis of blood samples taken at 2 weeks revealed that both fish oil and probucol lowered total plasma cholesterol by 30% compared with the CO group. In addition, fish oil supplementation caused a significant decrease in cholesterol contained in the VLDL fraction while probucol supplementation caused a significant lowered cholesterol in the HDL fraction. Analysis of mononuclear cell adhesion to the aortic endothelium in vivo revealed that, fish oil had no significant effect probucol reduced adhesion by 40%. The results of this study suggest that probucol, but not fish oil, may inhibit the initiation of lesion formation in the rat model of atherosclerosis.

n-3 fatty acid incorporation into LDL particles renders them more susceptible to oxidation in vitro but not necessarily more atherogenic in vivo.

The hypothesis that n-3 fatty acid incorporation into low-density lipoprotein (LDL) particles renders them more susceptible to oxidative modification and possibly more atherogenic was tested using two groups of female Yucatan miniature swine (10 animals per group) fed an atherogenic diet for 8 months. As a supplement to the atherogenic diet, the first group received a daily oral dose of the fish oil (FO) concentrate MaxEPA, rich in n-3 fatty acids, while the second group received the same dosage of a control oil (CO) low in n-3 fatty acids but with the same ratio of polyunsaturated to monounsaturated to saturated fatty acids as MaxEPA. At 8 months, the animals were killed and perfusion fixed, and all major vessels were removed for morphological assessment of atherosclerotic lesion area. Before fixation, blood samples were collected from all 20 pigs, and LDL (d = 1.019 to 1.063 g/mL) was separated from the plasma by ultracentrifugation. A series of in vitro oxidative modification reactions were carried out by incubating the LDL with a copper sulfate solution. The susceptibility of each LDL preparation to oxidation was determined by measuring both the formation of conjugated dienes and the relative mobility of each sample in an agarose gel. The incorporation of n-3 fatty acids into LDL particles decreased the lag phase by 30%, resulting in an increased mobility of FO-LDL (compared with CO-LDL) when incubated for 0.5 to 12 hours, but at longer incubation times (18 to 24 hours), the extent of modification between the two groups became equal.(ABSTRACT TRUNCATED AT 250 WORDS)