Search

Epidemiological studies indicate long-term risks of ionizing radiation on the heart, even at moderate doses. In this study, we investigated the inflammatory, thrombotic and fibrotic late responses of the heart after low-dose irradiation (IR) with specific emphasize on the dose rate. Hypercholesterolemic ApoE-deficient mice were sacrificed 3 and 6 months after total body irradiation (TBI) with 0.025, 0.05, 0.1, 0.5 or 2 Gy at low (1 mGy/min) or high dose rate (150 mGy/min). The expression of inflammatory and thrombotic markers was quantified in frozen heart sections (CD31, E-selectin, thrombomodulin, ICAM-1, VCAM-1, collagen IV, Thy-1, and CD45) and in plasma samples (IL6, KC, MCP-1, TNFα, INFγ, IL-1β, TGFβ, INFγ, IL-10, sICAM-1, sE-selectin, sVCAM-1 and fibrinogen) by fluorescence analysis and ELISA. We found that even very low irradiation doses induced adaptive late responses, such as increases of capillary density and changes in collagen IV and Thy-1 levels indicating compensatory regulation. Slight decreases of ICAM-1 levels and reduction of Thy 1 expression at 0.025-0.5 Gy indicate anti-inflammatory effects, whereas at the highest dose (2 Gy) increased VCAM-1 levels on the endocardium may represent a switch to a pro-inflammatory response. Plasma samples partially confirmed this pattern, showing a decrease of proinflammatory markers (sVCAM, sICAM) at 0.025-2.0 Gy. In contrast, an enhancement of MCP-1, TNFα and fibrinogen at 0.05-2.0 Gy indicated a proinflammatory and prothrombotic systemic response. Multivariate analysis also revealed significant age-dependent increases (KC, MCP-1, fibrinogen) and decreases (sICAM, sVCAM, sE-selectin) of plasma markers. This paper represents local and systemic effects of low-dose irradiation, including also age- and dose rate-dependent responses in the ApoE-/- mouse model. These insights in the multiple inflammatory/thrombotic effects caused by low-dose irradiation might facilitate an individual evaluation and intervention of radiation related, long-term side effects but also give important implications for low dose anti-inflammatory radiotherapy.

Angiotensin II favors the development of atherosclerosis. Our goal was to determine if foam cell formation increases angiotensin II generation by the endogenous renin-angiotensin system (RAS) and if endogenously produced angiotensin II promotes lipid accumulation in macrophages. Differentiated THP-1 cells were treated with acetylated low-density lipoproteins (ac-LDL), native LDL (n-LDL), or no LDL. Expression of RAS genes was assessed and angiotensin I/II levels were quantified in media and cell lysate. Ac-LDL increased angiotensin I/II levels and the angiotensin II/I ratio in cells and media after foam cell formation. Renin mRNA or activity did not change, but renin blockade completely inhibited the increase in angiotensin II. Angiotensinogen mRNA but not protein level was increased. Angiotensin-converting enzyme (ACE) and cathepsin G mRNA and activities were enhanced by ac-LDL. Inhibition of renin, ACE, or the angiotensin II receptor 1 (AT1-receptor) largely abolished cholesteryl ester formation in cells exposed to ac-LDL and decreased scavenger receptor A (SR-A) and acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT-1) protein levels. Inhibition of renin or the AT1-receptor in cells treated with oxidized LDL also decreased SR-A and ACAT-1 protein and foam cell formation. ac-LDL also increased angiotensin II by human peripheral blood monocyte-derived macrophages, whereas blockade of renin decreased cholesterol ester formation in these macrophages. These findings indicate that, during foam cell formation, angiotensin II generation by the endogenous RAS is stimulated and that endogenously generated angiotensin II is crucial for cholesterol ester accumulation in macrophages exposed to modified LDL.

Cathepsin G is a serine protease with a broad range of catalytic activities, including production of angiotensin II, degradation of extracellular matrix and cell-cell junctions, modulation of chemotactic responses, and induction of apoptosis. Cathepsin G mRNA expression is increased in human coronary atheroma vs. the normal vessel. To assess whether cathepsin G modulates atherosclerosis, cathepsin G knockout (Cstg(-/-)) mice were bred with apolipoprotein E knockout (Apoe(-/-)) mice to obtain Ctsg(+/-)Apoe(-/-) and Ctsg(+/+)Apoe(-/-) mice. Heterozygous cathepsin G deficiency led to a 70% decrease in cathepsin G activity in bone marrow cells, but this reduced activity did not impair generation of angiotensin II in bone marrow-derived macrophages (BMDM). Atherosclerotic lesions were compared in male Cstg(+/-)Apoe(-/-) and Cstg(+/+)Apoe(-/-) mice after 8 wk on a high-fat diet. Plasma cholesterol levels and cholesterol distribution within serum lipoprotein fractions did not differ between genotypes nor did the atherosclerotic lesion areas in either the aortic root or aortic arch. Cstg(+/-)Apoe(-/-) mice, however, showed a lower percentage of complex lesions within the aortic root and a smaller number of apoptotic cells compared with Cstg(+/+)Apoe(-/-) littermates. Furthermore, apoptotic Cstg(-/-) BMDM were more efficiently engulfed by phagocytic BMDM than were apoptotic Ctsg(+/+) BMDM. Thus cathepsin G activity may impair efferocytosis, which could lead to an accumulation of lesion-associated apoptotic cells and the accelerated progression of early atherosclerotic lesions to more complex lesions in Apoe(-/-) mice.

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.

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.

Objective: Naringenin is a citrus flavonoid that potently inhibits the assembly and secretion of apolipoprotein B100-containing lipoproteins in cultured hepatocytes and improves the dyslipidemia and insulin resistance in a mouse model of the metabolic syndrome. In the present study, we used low-density lipoprotein receptor-null mice fed a high-fat diet (Western, TD96125) to test the hypothesis that naringenin prevents atherosclerosis., Methods and Results: Three groups (chow, Western, and Western plus naringenin) were fed ad libitum for 6 months. The Western diet increased fasting plasma triglyceride (TG) (5-fold) and cholesterol (8-fold) levels compared with chow, whereas the addition of naringenin significantly decreased both lipids by 50%. The Western-fed mice developed extensive atherosclerosis in the aortic sinus because plaque area was increased by 10-fold compared with chow-fed animals. Quantitation of fat-soluble dye (Sudan IV)-stained aortas, prepared en face, revealed that Western-fed mice also had a 10-fold increase in plaque deposits throughout the arch and in the abdominal sections of the aorta, compared with chow. Atherosclerosis in both areas was significantly decreased by more than 70% in naringenin-treated mice. Consistent with quantitation of aortic lesions, the Western-fed mice had a significant 6-fold increase in cholesterol and a 4-fold increase in TG deposition in the aorta compared with chow-fed mice. Both were reduced more than 50% by naringenin. The Western diet induced extensive hepatic steatosis, with a 10-fold increase in both TG and cholesteryl ester mass compared with chow. The addition of naringenin decreased both liver TG and cholesteryl ester mass by 80%. The hyperinsulinemia and obesity that developed in Western-fed mice was normalized by naringenin to levels observed in chow-fed mice., Conclusions: These in vivo studies demonstrate that the citrus flavonoid naringenin ameliorates the dyslipidemia in Western-fed low-density lipoprotein receptor-null mice, leading to decreased atherosclerosis; and suggests a potential therapeutic strategy for the hyperlipidemia and increased risk of atherosclerosis associated with insulin resistance.

Aims: CD1d-restricted natural killer T (NKT) cells function by regulating numerous immune responses during innate and adaptive immunity. Depletion of all populations of CD1d-dependent NKT cells has been shown by several groups to reduce atherosclerosis in two different mouse models of the disease. In this study, we determined if removal of a single (V alpha 14) NKT cell population protects mice from the disease., Methods and Results: Targeted deletion of the J alpha 18 gene results in selective depletion of CD1d-dependent V alpha 14 NKT cells in C57BL/6 mice without affecting the population of other NKT, NK, and conventional T cells. Therefore, to study the effect of V alpha 14 NKT cell depletion on the progression of atherosclerosis, we examined the extent of lesion formation using paired littermate LDL receptor null mice that were either +/+ or -/- for the J alpha 18 gene following the feeding of these mice a cholesterol- and fat-enriched diet for 8 weeks. At the end of the study, we found no difference in either serum total- or lipoprotein-cholesterol distributions between groups. However, quantification of atherosclerosis revealed that V alpha 14 NKT cell deficiency significantly decreased lesion size in the aortic root (20-28%) and arch (28-38%) in both genders of mice. By coupling the techniques of laser capture microdissection with quantitative real-time RT-PCR, we found that expression of the proatherogenic cytokine interferon (IFN)-gamma was significantly reduced in lesions from J alpha 18-/- mice., Conclusion: This study is the first to identify a specific subpopulation of NKT cells that promotes atherosclerosis via a mechanism appearing to involve IFN-gamma expression.

Allograft coronary disease is the dominant cause of increased risk of death after cardiac transplantation. While the percutaneous insertion of stents is the most efficacious revascularization strategy for allograft coronary disease there is a high incidence of stent renarrowing. We developed a novel rabbit model of sex-mismatched allograft vascular disease as well as the response to stent implantation. In situ hybridization for the Y-chromosome was employed to detect male cells in the neointima of stented allograft, and the population of recipient derived neointimal cells was measured by quantitative polymerase chain reaction and characterized by immunohistochemistry. To demonstrate the participation of circulatory derived cells in stent neointima formation we infused ex vivo labeled peripheral blood mononuclear cells into native rabbit carotid arteries immediately after stenting. Fourteen days after stenting the neointima area was 58% greater in the stented vs. non-stented allograft segments (p = 0.02). Male cells were detected in the neointima of stented female-to-male allografts. Recipient-derived cells constituted 72.1+/-5.7% and 81.5+/-4.2% of neointimal cell population in the non-stented and stented segments, respectively and the corresponding proliferation rates were only 2.7+/-0.5% and 2.3+/-0.2%. Some of the recipient-derived neointimal cells were of endothelial lineage. The ex vivo tagged cells constituted 9.0+/-0.4% of the cells per high power field in the stent neointima 14 days after stenting. These experiments provide important quantitative data regarding the degree to which host-derived blood-borne cells contribute to neointima formation in allograft vasculopathy and the early response to stent implantation.

Objective: To determine whether expression of the human CETP transgene protects against diet-induced atherosclerosis in SR-BI deficient mice., Methods and Results: SR-BI deficient (-/-) mice were crossed with CETP transgenic (CETPtg) mice to produce a colony of SR-BI(-/-) x CETPtg mice in a C57Bl/6 background. Age and sex matched groups of genetically modified and wild-type C57Bl/6 mice were fed a high fat, high cholesterol diet for 22 weeks. In both wild-type and SR-BI(-/-) mice, expression of the CETP transgene reduced the cholesterol content and increased the density of lipoprotein particles in the HDL density range. In SR-BI(-/-) x CETPtg mice, CETP activity inversely correlated with total plasma cholesterol levels and shifted the buoyant HDL typical of SR-BI deficiency toward a more normal density HDL particle. Atherosclerosis at the level of the aortic arch was evident in both male and female SR-BI deficient mice but occurred to a greater extent in the females. Expression of CETP markedly attenuated the development of atherosclerosis in SR-BI deficient mice fed an atherogenic diet (P<0.003)., Conclusions: Expression of the human CETP transgene protects SR-BI deficient mice from atherosclerosis, consistent with a role for CETP in remodeling HDL and providing an alternative pathway for the selective uptake of HDL-CE by the liver.

Endocytosis of LDL and modified LDL represents regulated and unregulated cholesterol delivery to macrophages. To elucidate the mechanisms of cellular cholesterol transport and egress under both conditions, various primary macrophages were labeled and loaded with cholesterol or cholesteryl ester from LDL or acetylated low density lipoprotein (AcLDL), and the cellular cholesterol traffic pathways were examined. Confocal microscopy using fluorescently labeled 3,3'-dioctyldecyloxacarbocyanine perchlorate-labeled LDL and 1,1'-dioctyldecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate-labeled AcLDL demonstrated their discrete traffic pathways and accumulation in distinct endosomes. ABCA1-mediated cholesterol efflux to apolipoprotein A-I (apoA-I) was much greater for AcLDL-loaded macrophages compared with LDL. Treatment with the liver X receptor ligand 22-OH increased efflux to apoA-I in AcLDL-loaded but not LDL-loaded cells. In contrast, at a level equivalent to AcLDL, LDL-derived cholesterol was preferentially effluxed to HDL, in keeping with increased ABCG1. In vivo studies of reverse cholesterol transport (RCT) from cholesterol-labeled macrophages injected intraperitoneally demonstrated that LDL-derived cholesterol was more efficiently transported to the liver and secreted into bile than AcLDL-derived cholesterol. This indicates a greater efficiency of HDL than lipid-poor apoA-I in interstitial fluid in controlling in vivo RCT. These assays, taken together, emphasize the importance of mediators of diffusional cholesterol efflux in RCT.

Objective: CD133 may be the most specific marker of endothelial progenitor cells (EPCs), which are thought to be largely confined to the bone marrow milieu. This study reports on the phenotypic characterization and functional analysis of human CD133+ cells and their generation from cells in the peripheral circulation., Methods: Adult human CD133+ and CD133- cells were isolated from peripheral blood mononuclear cells, and the generation of CD133+ cells in culture was attempted using different culture combinations. The phenotypic, migratory, adhesive, and angiogenic properties of the native and generated populations were investigated., Results: In adherent and in suspension culture systems, CD133+ cells also expressing CD34 and VEGFR-2 were successfully derived from a previously CD133- population. The migratory potential of CD133+ cells was enhanced by the presence of the CD133- cells. Also, the CD133+ cells derived from the CD133- cells demonstrated improved adhesion to extracellular matrix and endothelial monolayer substrates, and their contribution to in vitro angiogenesis was enhanced compared to freshly isolated CD133+ cells., Conclusions: These results demonstrate a source of blood CD133+ cells other than direct mobilization from the bone marrow. Cellular interaction was observed between fractions, with CD133+ cells showing better in vitro function in the presence of CD133- cells. These findings provide a novel source for CD133+ cells and a rationale for the investigation of angiogenic cell recruitment or delivery strategies involving more than one cell type at ischemic sites.

Epithelial sodium channels (ENaC) are important for regulating sodium transport across epithelia. Functional studies indicate that neural mechanisms acting through mineralocorticoid receptors (MR) and sodium channels (presumably ENaC) are crucial to the development of sympathoexcitation and hypertension in experimental models of salt-sensitive hypertension. However, expression and localization of the ENaC in cardiovascular regulatory centers of the brain have not yet been studied. RT-PCR and immunohistochemistry were performed to study ENaC and MR expression at the mRNA and protein levels, respectively. Both mRNA and protein for alpha-, beta-, and gamma-ENaC subunits and MR were found to be expressed in the rat brain. All three ENaC subunits and MR were present in the supraoptic nucleus, magnocellular paraventricular nucleus, hippocampus, choroid plexus, ependyma, and brain blood vessels, suggesting the presence of multimeric channels and possible regulation by mineralocorticoids. In most cortical areas, thalamus, amygdala, and suprachiasmatic nucleus, notable expression of gamma-ENaC was undetectable, whereas alpha- and beta-ENaC were abundantly expressed pointing to the possibility of a heterogeneous population of channels. The findings suggest that stoichiometrically different populations of ENaC may be present in both epithelial and neural components in the brain, which may contribute to regulation of cerebrospinal fluid and interstitial Na+ concentration as well as neuronal excitation.

Objective: Natural killer (NK) cells are a key component of innate immunity. Despite being identified in human and mouse atherosclerotic lesions, the role of NK cells in the disease process in unknown. To determine this role, we created chimeric atherosclerosis-susceptible low-density lipoprotein (LDL) receptor null (ldl-r-/-) mice that were deficient in functional NK cells through expression of a transgene encoding for Ly49A., Methods and Results: Bone marrow cells from Ly49A transgenic and nontransgenic littermates were used to repopulate the hematopoietic system of lethally-irradiated female ldl-r-/- mice. After a recovery period to permit sufficient engraftment, mice were placed on a diet enriched in saturated fat and cholesterol. After 8 weeks, there was no difference in either serum total cholesterol concentrations or lipoprotein cholesterol distribution in mice repopulated with nontransgenic versus Ly49A transgenic marrow cells. Using immunohistochemistry, we detected NK cells in atherosclerotic lesions of both groups of mice. However, deficiency of functional NK cells significantly reduced the size of atherosclerosis by 70% (P=0.0002) in cross-sectional analysis of the aortic root and by 38% (P=0.004) in en face analysis of the intimal surface of the aortic arch., Conclusions: These studies demonstrate that NK cells infiltrate the vessel wall and promote atherosclerotic lesion development.

This review discusses the application-side of using mice as an animal model of atherosclerosis, and is directed towards the researcher new to using mice to perform atherosclerosis studies. Although this review will comment on many of the current mouse models that are available, noting their strengths and weaknesses, the majority of this review is relevant to planning experiments involving either apolipoprotein (apo) E deficient or low-density lipoprotein (LDL) receptor deficient mice. Subject matter covered includes a description of the types of lesions expected to form in apoE deficient and LDL receptor deficient mice, the age of the mouse when these various types of lesion are expected to form, and finally, a description of the most popular methods used to perform both biochemical and morphometric analysis of atherosclerotic lesions.

Class A scavenger receptors (SR-A) have been implicated in the atherogenic process, although there have been conflicting reports as to their specific effect on the development of lesions. In part, this discord may arise because of the variable contribution of SR-A in the several cell types known to express this protein. To determine the effects of macrophage-specific SR-A expression in the atherogenic process, transgenic mice were created using the chicken lysozyme (lyso) promoter to drive expression of bovine SR-A (bSR-A). To express this gene in an atherosclerosis-susceptible strain, bone marrow cells from transgenic and non-transgenic littermates were used to repopulate lethally-irradiated female LDL receptor (LDLr)(-/-) mice. Following hematopoietic engraftment, mice were placed on a diet enriched in saturated fat and cholesterol. After 8 weeks, there was a modest, but statistically significant reduction in serum total cholesterol in LDLr(-/-) mice repopulated with lyso-bSR-A transgenic cells, due to decreased LDL-cholesterol. The extent of atherosclerosis was reduced in both cross-sectional analysis of the aortic root and en face analysis of the intimal surface of the aortic arch. In addition to changes in atherosclerosis, lyso-bSR-A repopulated LDLr(-/-) mice had a marked increase (3.6x) in spleen weights and a disruption of spleen white pulp formation. Therefore, macrophage-specific overexpression of SR-A resulted in reduced atherosclerosis in two vascular beds, reduced serum cholesterol concentrations, and changed the morphology of the spleen.

We have previously shown that interferon-gamma (IFN-gamma) is a potent enhancer of atherogenesis. Interleukin-18 (IL-18) promotes inflammatory responses through release of IFN-gamma, although it can also exert direct actions on other inflammatory mediators. In this present study, we determined the effects of IL-18 on atherogenesis and the role of IFN-gamma in this response. Male apolipoprotein E(-/-) mice (apoE(-/-); aged 16 weeks, n=10/group) were fed a normal diet and injected intraperitoneally for 30 days with either recombinant IL-18 (30 ng/g/day) or saline. Atherosclerotic lesion size was quantified in 2 vascular beds: the ascending aorta and the aortic arch. IL-18 administration did not affect serum cholesterol concentrations or lipoprotein-cholesterol distribution; however, exogenous IL-18 administration increased lesion size 2-fold in both the ascending aorta (50 642 +/- 12 515 versus 112 399 +/- 13 227 microm(2) P=0.004; saline versus IL-18 groups, respectively) and the aortic arch (3.1 +/- 0.3% versus 6.2 +/- 0.9% area, P=0.006). Exogenous IL-18 promoted a 4-fold increase in the number of lesion-associated T lymphocytes (11 +/- 3 versus 50 +/- 5 cells; P<0.0001) and cells expressing major histocompatability complex class II (9 +/- 3 versus 40 +/- 6 cells; P=0.0002). To determine the role of IFN-gamma production in this response, exogenous IL-18 was administered to apoE(-/-) mice that were IFN-gamma deficient. These studies demonstrated that lack of endogenous IFN-gamma ablated the effects of IL-18 on atherosclerosis. Therefore, these data strongly implicates IL-18 in the atherogenic process and suggests that IL-18 increases lesion development through enhancement of an inflammatory response involving an IFN-gamma-dependent mechanism. The full text of this article is available at http://www.circresaha.org.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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)