Faculty Bibliography

OBJECTIVE: Acyl-CoA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters in plaque foam cells. Complete deficiency of macrophage ACAT has been shown to increase atherosclerosis in hypercholesterolemic mice because of cytotoxicity from free cholesterol accumulation, whereas we previously showed that partial ACAT inhibition by Fujirebio compound F1394 decreased early atherosclerosis development. In this report, we tested F1394 effects on preestablished, advanced lesions of apolipoprotein-E-deficient mice. METHODS AND RESULTS: Apolipoprotein-E-deficient mice on Western diet for 14 weeks developed advanced plaques, and were either euthanized (Baseline), or continued on Western diet with or without F1394 and euthanized after 14 more weeks. F1394 was not associated with systemic toxicity. Compared with the baseline group, lesion size progressed in both groups; however, F1394 significantly retarded plaque progression and reduced plaque macrophage, free and esterified cholesterol, and tissue factor contents compared with the untreated group. Apoptosis of plaque cells was not increased, consistent with the decrease in lesional free cholesterol. There was no increase in plaque necrosis and unimpaired efferocytosis (phagocytic clearance of apoptotic cells). The effects of F1394 were independent of changes in plasma cholesterol levels. CONCLUSIONS: Partial ACAT inhibition by F1394 lowered plaque cholesterol content and had other antiatherogenic effects in advanced lesions in apolipoprotein-E-deficient mice without overt systemic or plaque toxicity, suggesting the continued potential of ACAT inhibition for the clinical treatment of atherosclerosis, in spite of recent trial data.

Our lab has previously shown in a mouse model that normalization of a low HDL level achieves atherosclerotic plaque regression. This included the shift from a pro ("M1") to an anti-inflammatory ("M2") phenotypic state of plaque macrophages. Whether HDL can directly cause this phenotypic change and, if so, what the signaling mechanism is, were explored in the present studies. Murine primary macrophages treated with HDL showed increased gene expression for the M2 markers Arginase-1 (Arg-1) and Fizz-1, which are classically induced by IL-4. HDL was able to potentiate the IL-4-induced changes in Arg-1, and tended to do the same for Fizz-1, while suppressing the expression of inflammatory genes in response to IFNgamma. The effects of either IL-4 or HDL were suppressed when macrophages were from STAT6(-/-) mice, but inhibitor studies suggested differential utilization of JAK isoforms by IL-4 and HDL to activate STAT6 by phosphorylation. Overall, our results describe a new function of HDL, namely its ability to directly enrich macrophages in markers of the M2, anti-inflammatory, state in a process requiring STAT6.

Coronary artery disease, resulting from atherosclerosis, is the leading cause of death in the Western world. Most previous studies have subjected atherosclerotic arteries, a tissue of mixed cellular composition, to homogenization in order to identify the factors in plaque development, thereby obscuring information relevant to specific cell types. Because macrophage foam cells are critical mediators in atherosclerotic plaque advancement, we reasoned that performing gene analysis on those cells would provide specific insight in novel regulatory factors and potential therapeutic targets. We demonstrated for the first time in vascular biology that foam cell-specific RNA can be isolated by laser capture microdissection (LCM) of plaques. As expected, compared to whole tissue, a significant enrichment in foam cell-specific RNA transcripts was observed. Furthermore, because regression of atherosclerosis is a tantalizing clinical goal, we developed and reported a transplantation-based mouse model. This involved allowing plaques to form in apoE-/- mice and then changing the plaque's plasma environment from hyperlipidemia to normolipidemia. Under those conditions, rapid regression ensued in a process involving emigration of plaque foam cells to regional and systemic lymph nodes. Using LCM, we were able to show that under regression conditions, there was decreased expression in foam cells of inflammatory genes, but an up-regulation of cholesterol efflux genes. Interestingly, we also found that increased expression of chemokine receptor CCR7, a known factor in dendritic cell migration, was required for regression. In conclusion, the LCM methods described in this chapter, which have already lead to a number of striking findings, will likely further facilitate the study of cell type-specific gene expression in animal and human plaques during various stages of atherosclerosis, and after genetic, pharmacologic, and environmental perturbations.

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Galphai signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14(+) human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators.

Both in humans and animal models, an acute increase in plasma insulin levels, typically following meals, leads to transient depression of hepatic secretion of very low density lipoproteins (VLDL). One contributing mechanism for the decrease in VLDL secretion is enhanced degradation of apolipoprotein B100 (apoB100), which is required for VLDL formation. Unlike the degradation of nascent apoB100, which occurs in the endoplasmic reticulum (ER), insulin-stimulated apoB100 degradation occurs post-ER and is inhibited by pan-phosphatidylinositol (PI)3-kinase inhibitors. It is unclear, however, which of the three classes of PI3-kinases is required for insulin-stimulated apoB100 degradation, as well as the proteolytic machinery underlying this response. Class III PI3-kinase is not activated by insulin, but the other two classes are. By using a class I-specific inhibitor and siRNA to the major class II isoform in liver, we now show that it is class II PI3-kinase that is required for insulin-stimulated apoB100 degradation in primary mouse hepatocytes. Because the insulin-stimulated process resembles other examples of apoB100 post-ER proteolysis mediated by autophagy, we hypothesized that the effects of insulin in autophagy-deficient mouse primary hepatocytes would be attenuated. Indeed, apoB100 degradation in response to insulin was significantly impaired in two types of autophagy-deficient hepatocytes. Together, our data demonstrate that insulin-stimulated apoB100 degradation in the liver requires both class II PI3-kinase activity and autophagy.

Rationale: Statins have been shown to have anti-inflammatory pleiotropic effects in experimental settings. However, the bioavailability of orally administered statins is poor, which limits the direct therapeutic effects on atherosclerotic plaques. We have developed a simvastatin loaded high-density lipoprotein nanoparticle platform ([S]-rHDL), with the similar properties to natural HDL (A). The formulation has potent anti-inflammatory effects in vitro (S-A), produces significantly higher therapeutic effects in apolipoprotein E knockout (ApoE KO) mice than oral simvastatin or rHDL treatment, and induces rapid regression when administered at a high dose (S-B). The purpose of the current study is to understand the mechanism of action by which this formulation exerts its potent antiinflammatory effects. Methods and Results: To investigate the protecting effects of [S]-rHDL on encapsulated simvastatin in blood, the nanoparticles were incubated in mouse serum and intact simvastatin was measured over time with HPLC. [S]-rHDL drastically protected the encapsulated simvastatin (B). To evaluate the in vivo targeting of atherosclerotic plaques, [S]-rHDL was additionally labeled with Gd-DPTA-lipids and Cy5.5-DMPE, and intravenously administered in ApoE KO mice. In vivo T1-weighted magnetic resonance imaging (MRI) revealed [S]-rHDL accumulation in atherosclerotic plaques in the abdominal aortas (C), while ex vivo nearinfrared fluorescence imaging (NIRF) of excised aortas demonstrated pronounced accumulation in plaque rich lesions such as the aortic roots and arches (D). To investigate the cellular targeting specificity of [S]-rHDL, flow cytometry and fluorescence microscopy were used to analyze the blood, spleen, and atherosclerotic plaques. [S]-rHDL was preferentially taken up by monocytes and macrophages in the blood and spleen (E), but not by other leukocytes (S-D). It also extensively accumulated in atherosclerotic plaques, and was taken up by plaque macrophages (F and S-C). Altogether these data suppor!

Although high high-density lipoprotein (HDL)-cholesterol levels are associated with decreased cardiovascular risk in epidemiological studies, recent genetic and pharmacological findings have raised doubts about the beneficial effects of HDL. Raising HDL levels in animal models by infusion or overexpression of apolipoprotein A-I has shown clear vascular improvements, such as delayed atherosclerotic lesion progression and accelerated lesion regression, along with increased reverse cholesterol transport. Inflammation and other factors, such as myeloperoxidase-mediated oxidation, can impair HDL production and HDL function, with regard to its reverse cholesterol transport, antioxidant, and anti-inflammatory activities. Thus, tests of HDL function, which have not yet been developed as routine diagnostic assays, may prove useful and be a better predictor of cardiovascular risk than HDL-cholesterol levels.

Rationale: Although statins have been shown to have anti-inflammatory pleiotropic effects in experimental studies, the poor plaque targeting of orally administered statins limits their direct anti-inflammatory therapeutic effect. To that end, we have developed a simvastatin loaded high-density lipoprotein nanoparticle ([S]-rHDL), which has an improved plaque bioavailability and therefore exerts a higher therapeutic effect in apolipoprotein E knockout (ApoE KO) mice than orally administered simvastatin. The purpose of the current study is to understand the mechanism of this potent anti-inflammatory effect. Methods and Results: [S]-rHDL was found to specifically target plaque macrophages by fluorescence microscopy (a). To investigate the targeting efficiency of [S]-rHDL to monocytes/macrophages, we intravenously injected [S]-rHDL in ApoE KO mice (n=3/time point) and analyzed the cells from aortas and blood by flow cytometry. [S]-rHDL was found to target macrophages and monocytes in aortas (b), and to target inflammatory Gr-1hi monocytes more efficiently than anti-inflammatory Gr-1lo monocytes in blood (c). Last, laser capture microdissection was used to isolate plaque macrophages (n=7), and quantitative RT-PCR was used to measure their mRNA level of TNF-alpha, the hallmark of macrophage inflammation, which was significantly reduced (d). All the above data support our hypothesis that [S]-rHDL acts on inflammatory monocytes and plaque macrophages and thereby exerts a strong anti-inflammatory effect on atherosclerotic plaque. Conclusion: In ApoE KO mice, [S]-rHDL specifically targets plaque macrophages. [S]-rHDL also locally acts on macrophages in plaque and preferentially targets pro-inflammatory monocytes in blood, which results in a strong anti-inflammatory effect. This nanotherapy may represent a potent addition to the current standard of care for atherosclerosis patients

Up to 1 in 3000 individuals in the United States have alpha-1 antitrypsin deficiency, and the most common cause of this disease is homozygosity for the antitrypsin-Z variant (ATZ). ATZ is inefficiently secreted, resulting in protein deficiency in the lungs and toxic polymer accumulation in the liver. However, only a subset of patients suffer from liver disease, suggesting that genetic factors predispose individuals to liver disease. To identify candidate factors, we developed a yeast ATZ expression system that recapitulates key features of the disease-causing protein. We then adapted this system to screen the yeast deletion mutant collection to identify conserved genes that affect ATZ secretion and thus may modify the risk for developing liver disease. The results of the screen and associated assays indicate that ATZ is degraded in the vacuole after being routed from the Golgi. In fact, one of the strongest hits from our screen was Vps10, which can serve as a receptor for the delivery of aberrant proteins to the vacuole. Because genome-wide association studies implicate the human Vps10 homolog, sortilin, in cardiovascular disease, and because hepatic cell lines that stably express wild-type or mutant sortilin were recently established, we examined whether ATZ levels and secretion are affected by sortilin. As hypothesized, sortilin function impacts the levels of secreted ATZ in mammalian cells. This study represents the first genome-wide screen for factors that modulate ATZ secretion and has led to the identification of a gene that may modify disease severity or presentation in individuals with ATZ-associated liver disease.