Faculty Bibliography

Mouse aortic smooth muscle cells (SMCs) were loaded for 72 h with cholesterol by using cholesterol:methyl-beta-cyclodextrin complexes, leading to approximately 2-fold and approximately 10-fold increases in the contents of total cholesterol and cholesteryl ester, respectively. Foam-cell formation was demonstrated by accumulation of intracellular, Oil Red O-stained lipid droplets. Immunostaining showed decreased protein levels of smooth muscle alpha-actin and alpha-tropomyosin and increased levels of macrophage markers CD68 and Mac-2 antigen. Quantitative real-time RT-PCR revealed that after cholesterol loading, the expression of SMC-related genes alpha-actin, alpha-tropomyosin, myosin heavy chain, and calponin H1 decreased (to 11.5 +/- 0.5%, 29.3 +/- 1.4%, 23.8 +/- 1.4%, and 3.8 +/- 0.5% of unloaded cells, respectively; P < 0.05 for all), whereas expression of macrophage-related genes CD68, Mac-2, and ABCA1 mRNA increased (to 709 +/- 84%, 330 +/- 11%, and 207 +/- 13% of unloaded cells, respectively; P < 0.05 for all), thereby demonstrating that the protein changes were regulated at the mRNA level. Furthermore, these changes were accompanied by a gain in macrophage-like function as assessed by phagocytotic activity. Expression of vascular cell adhesion molecule 1 and monocyte chemoattractant protein 1, known responders to inflammation, were not changed. In conclusion, cholesterol loading of SMC causes phenotypic changes regulated at the mRNA level that result in a transdifferentiation to a macrophage-like state. This finding suggests that not all foam cells in lesions may have a macrophage origin, despite what is indicated by immunostaining for macrophage-related markers. Furthermore, inflammatory changes in foam cells observed in vivo may not be simple consequences of cholesterol accumulation.

Excess cellular cholesterol induces apoptosis in macrophages, an event likely to promote progression of atherosclerosis. The cellular mechanism of cholesterol-induced apoptosis is unknown but had previously been thought to involve the plasma membrane. Here we report that the unfolded protein response (UPR) in the endoplasmic reticulum is activated in cholesterol-loaded macrophages, resulting in expression of the cell death effector CHOP. Cholesterol loading depletes endoplasmic reticulum calcium stores, an event known to induce the UPR. Furthermore, endoplasmic reticulum calcium depletion, the UPR, caspase-3 activation and apoptosis are markedly inhibited by selective inhibition of cholesterol trafficking to the endoplasmic reticulum, and Chop(-/-) macrophages are protected from cholesterol-induced apoptosis. We propose that cholesterol trafficking to endoplasmic reticulum membranes, resulting in activation of the CHOP arm of the UPR, is the key signalling step in cholesterol-induced apoptosis in macrophages

Apolipoprotein B100 (apoB) is a large (520-kDa) complex secretory protein; its secretion is regulated posttranscriptionally by several degradation pathways. The best described of these degradative processes is co-translational ubiquitinylation and proteasomal degradation of nascent apoB, involving the 70- and 90-kDa heat shock proteins and the multiple components of the proteasomal pathway. Ubiquitinylation involves several proteins, including ligases called E3s, that coordinate the covalent binding of ubiquitin to target proteins. The recent discovery that tumor autocrine motility factor receptor, also known as gp78, is an endoplasmic reticulum (ER)-associated E3, raised the possibility that this E3 might be involved in the ER-associated degradation of nascent apoB. In a series of experiments in HepG2 cells, we demonstrated that overexpression of gp78 was sufficient for increased ubiquitinylation and proteasomal degradation of apoB, with reduced secretion of apoB-lipoproteins. This action of gp78 was specific: overexpression of the protein did not affect secretion of either albumin or apolipoprotein AI. Furthermore, overexpression of a cytosolic E3, Itch, had no effect on apoB secretion. Finally, using an in vitro translation system, we demonstrated that gp78 led to increased ubiquitinylation and proteasomal degradation of apoB48. Together, these results indicate that an ER-associated protein, gp78, is a bona fide E3 ligase in the apoB ER-associated degradation pathway

Hyperglycemia derived advanced glycation endproducts (AGE) have been implicated in diabetic atherosclerosis (AS) but the role of exogenous (dietary) AGE in the development of this serious complication is not known. This study evaluates the influence of diet-related AGE on AS in genetically hypercholesterolemic apolipoprotein E-deficient (apoE(-/-)), streptozotocin-induced diabetic mice. Diabetic and non-diabetic apoE(-/-) mice (6-8 weeks old) were randomized into either a standard AIN-93G chow (AGE 12,500+/-700 U/mg, termed high-AGE diet, H-AGE), or the same chow having four to fivefold lower AGE level (L-AGE: 2,700+/-830 U/mg) based on ELISA. After 2 months of diabetes, compared to the diabetic mice fed standard (H-AGE) diet, the AS lesions at the aortic root of the L-AGE group were >50% smaller (0.17+/-0.03 vs. 0.31+/-0.05 mm(2), P<0.05). Serum AGE were lower in the diabetic L-AGE than in the H-AGE mice (by approximately 53%) (P<0.00001), as were in the non-diabetic L-AGE vs. H-AGE groups (P<0.05). No diet-related changes were noted in plasma glucose, triglycerides, or plasma cholesterol. Immunohistochemical comparisons showed markedly suppressed tissue AGE, AGE-Receptor-1, -2 and RAGE expression, reduced numbers of inflammatory cells, tissue factor, vascular cell adhesion molecule-1 and MCP-1 in the L-AGE diabetic group. The findings are supportive of an important link between dietary intake of pre-formed glycoxidation products, tissue-incorporated AGE, and diabetes-accelerated AS. The marked anti-atherogenic effects of an AGE-restricted diet in this model may provide the basis for relevant clinical studies

BACKGROUND: Syngeneic heterotopic transplantation of segments of descending thoracic aortas containing atherosclerotic lesions from hypercholesterolemic mice into normocholesterolemic recipients has been useful for studies on plaque regression and stabilization. Because lesion development is more rapid and exuberant in the aortic arch, a technique of transplantation of the mouse aortic arch was developed. MATERIALS AND METHODS: C57BL/6, apoE-deficient (apoE-/-) (hypercholesterolemic) mice were fed a Western diet for 22 weeks and used as donors of aortic-arch segments containing atherosclerotic lesions. Twenty syngeneic transplants were performed on age-matched wild-type (normocholesterolemic) mice. Aortic arches containing atherosclerotic lesions were implanted on the abdominal aorta of recipient mice by end-to-side microsurgical anastomosis. Two weeks after transplantation, grafts were noninvasively imaged in vivo by magnetic resonance (MR) microscopy. Grafts harvested four weeks after transplantation were submitted for histological examination. RESULTS: All recipients survived the entire follow-up period (1 month) without complications. Duration of recipient procedure ranged from 90 to 120 (mean, 105) min; aortic clamping time varied from 45 to 60 min. In vivo MR microscopy demonstrated patency of the grafts and wall thickening that corresponded to the preexisting atherosclerotic lesions. Histology confirmed patency and atherosclerotic thickening of the grafts, and showed no evidence of acute tissue damage. CONCLUSIONS: Syngeneic transplantation of the aortic arch in mice represents a useful alternative model for studies on morphology, imaging, and mechanisms of atherosclerosis. The curvature of the aortic arch is preserved after implantation onto the abdominal aorta, providing clear landmarks for noninvasive assessment using MR

BACKGROUND: LDL receptor-deficient 'apolipoprotein (apo)-B100-only' mice (Ldlr-/-Apob100/100 have elevated LDL cholesterol levels on a chow diet and develop severe aortic atherosclerosis. We hypothesized that both the hypercholesterolemia and the susceptibility to atherosclerosis could be eliminated by switching off hepatic lipoprotein production. METHODS AND RESULTS: We bred Ldlr-/-Apob100/100 mice that were homozygous for a conditional allele for Mttp (the gene for microsomal triglyceride transfer protein) and the inducible Mx1-Cre transgene. In these animals, which we called 'Reversa mice,' the hypercholesterolemia could be reversed, without modifying the diet or initiating a hypolipidemic drug, by the transient induction of Cre expression in the liver. After Cre induction, hepatic Mttp expression was virtually eliminated (as judged by quantitative real-time PCR), hepatic lipoprotein secretion was abolished (as judged by electron microscopy), and LDLs were virtually eliminated from the plasma. Intestinal lipoprotein production was unaffected. In mice fed a chow diet, Cre induction reduced plasma cholesterol levels from 233.9+/-46.0 to 37.2+/-6.5 mg/dL. In mice fed a high-fat diet, cholesterol levels fell from 525.7+/-32.2 to 100.6+/-14.3 mg/dL. The elimination of hepatic lipoprotein production completely prevented both the development of atherosclerosis and the changes in gene expression that accompany atherogenesis. CONCLUSIONS: We developed mice in which hypercholesterolemia can be reversed with a genetic switch. These mice will be useful for understanding gene-expression changes that accompany the reversal of hypercholesterolemia and atherosclerosis

In vivo, cardiac-gated, black-blood, and ex vivo magnetic resonance microscopy (MRM) images of the aortic root, and histopathology data were obtained from 12 transgenic and wild-type (WT) mice. MRM was performed using a black-blood imaging spin-echo sequence with upstream and downstream in-flow saturation pulses to obtain aortic root images in three contrast techniques: proton density-weighted (PDW), T(1)- (T(1)W), and T(2)-weighted (T(2)W). Aortic wall thickness and area were measured and correlated with histopathology data (R > 0.90). Ex vivo lesion components (lipid core, fibrous tissue, and cell tissue) were identified and characterized by differing image contrast in PDW, T(1)W, and T(2)W MRM, and by histopathology. The differences between WT and transgenic mice for maximal wall thickness and area were statistically significant (P < 0.05). This study demonstrates the feasibility of in vivo murine aortic root lesion assessment and ex vivo plaque characterization by MRM

PURPOSE: To test the ability of serial, in vivo magnetic resonance microscopy (MRM) to detect the development of atherosclerosis and quantify its progression in apolipoprotein E-deficient mice. MATERIALS AND METHODS: The abdominal aortae of six ApoE(-/-) and three wild-type (WT) control mice were imaged by MRM at 9.4T. Proton density weighted images were obtained (TR = 2000, TE = 9 msec) using four signal averages. The image resolution was 109 x 109 x 500 microm(3). The six ApoE(-/-) mice underwent serial MRM three to five times over a period < or = 44 weeks. Multiple, anatomically aligned MRM slices (N = 6-11 per time point, total 202) were compared serially in each animal. RESULTS: The abdominal aorta remained free of atherosclerosis until 20 weeks of age but thereafter, atherosclerosis was identified in all ApoE(-/-) mice (P < 0.05 to P < 0.001), but no WT controls. Lesion progression was accompanied by positive remodeling in which atherosclerosis within the aortic wall was accommodated by an increase in total cross sectional area (P < 0.01), while lumen area was unchanged. CONCLUSION: Serial MRM demonstrated the development and progression of atherosclerosis in mouse aorta. Importantly, progression of atherosclerosis could be identified within individual animals. By following the same aortic lesions over time, MRM demonstrated that progression of atherosclerosis in mice is associated with positive arterial remodeling

Fatty acids of varying lengths and saturation differentially affect plasma apolipoprotein B-100 (apoB-100) levels. To identify mechanisms at the level of production, rat hepatoma cells, McA-RH7777, were incubated with [(35)S]methionine and either fatty acid-BSA complexes or BSA alone. There were increases in labeled apoB-100 secretion with saturated fatty acids palmitic and myristic (MA) (153 +/- 20% and 165 +/- 11%, respectively, relative to BSA). Incubation with polyunsaturated docosahexaenoic acid (DHA) decreased secretion to 26 +/- 2.0%, while monounsaturated oleic acid (OA) did not change it. In pulse-chase studies, MA treatment resulted in reduced apoB-100 degradation, in agreement with its promotion of secretion. In triglyceride (TG) studies, synthesis was stimulated equally by OA, MA, and DHA, but TG secretion was relatively decreased with MA and DHA. With OA, the majority of newly secreted apoB100-lipoproteins was d < or = 1.006, but with MA, they were much denser (1.063 < d). Furthermore, the relative recruitment of newly synthesized TG to lipoproteins was impaired with MA. We conclude that mechanisms for effects of specific dietary fatty acids on plasma lipoprotein levels may include changes in hepatic production. In turn, hepatic production may be regulated by specific fatty acids at the steps of apoB-100 degradation and the recruitment of nascent TG to lipoprotein particles

OBJECTIVE: Monocyte chemoattractant protein (MCP)-1 is a proatherogenic factor that is responsible for approximately 60% of plaque macrophages in mouse models of atherosclerosis. We investigated whether lysophosphatidylcholine (LPC), enriched in oxidized low density lipoprotein, can modulate the expression of MCP-1 in arterial wall cells. METHODS AND RESULTS: LPC induced a 3-fold increase in MCP-1 mRNA in rat vascular smooth muscle cells (VSMCs) in a time- and dose-dependent manner. Nuclear runon analysis showed that this increase was attributable to increased MCP-1 gene transcription. There was a 2-fold increase in MCP-1 protein in the conditioned media of cells treated with LPC. LPC-associated increases of MCP-1 mRNA and protein were similar to those produced by platelet-derived growth factor-BB, a known inducer of MCP-1. Analyses of the MCP-1 promoter in transiently transfected VSMCs indicated an LPC-responsive element(s) between base pairs -146 and -261 (relative to transcription initiation). Further studies suggested that LPC-induced MCP-1 expression partially involves mitogen-activated protein kinase/extracellular signal-regulated kinase, a tyrosine kinase(s), and (to a lesser extent) protein kinase C but not the activation of the platelet-derived growth factor receptor. CONCLUSIONS: LPC stimulates MCP-1 expression at the transcriptional level in VSMCs, suggesting a molecular mechanism by which LPC contributes to the atherogenicity of oxidized low density lipoprotein