Faculty Bibliography

Tumor-associated macrophages (TAMs) are increasingly investigated in cancer immunology and are considered a promising target for better and tailored treatment of malignant growth. Although TAMs also have high diagnostic and prognostic value, TAM imaging still remains largely unexplored. Here, we describe the development of reconstituted high-density lipoprotein (rHDL)-facilitated TAM PET imaging in a breast cancer model. METHODS: Radiolabeled rHDL nanoparticles incorporating the long-lived positron-emitting nuclide (89)Zr were developed using 2 different approaches. The nanoparticles were composed of phospholipids and apolipoprotein A-I (apoA-I) in a 2.5:1 weight ratio. (89)Zr was complexed with deferoxamine (also known as desferrioxamine B, desferoxamine B), conjugated either to a phospholipid or to apoA-I to generate (89)Zr-PL-HDL and (89)Zr-AI-HDL, respectively. In vivo evaluation was performed in an orthotopic mouse model of breast cancer and included pharmacokinetic analysis, biodistribution studies, and PET imaging. Ex vivo histologic analysis of tumor tissues to assess regional distribution of (89)Zr radioactivity was also performed. Fluorescent analogs of the radiolabeled agents were used to determine cell-targeting specificity using flow cytometry. RESULTS: The phospholipid- and apoA-I-labeled rHDL were produced at 79% +/- 13% (n = 6) and 94% +/- 6% (n = 6) radiochemical yield, respectively, with excellent radiochemical purity (>99%). Intravenous administration of both probes resulted in high tumor radioactivity accumulation (16.5 +/- 2.8 and 8.6 +/- 1.3 percentage injected dose per gram for apoA-I- and phospholipid-labeled rHDL, respectively) at 24 h after injection. Histologic analysis showed good colocalization of radioactivity with TAM-rich areas in tumor sections. Flow cytometry revealed high specificity of rHDL for TAMs, which had the highest uptake per cell (6.8-fold higher than tumor cells for both DiO@Zr-PL-HDL and DiO@Zr-AI-HDL) and accounted for 40.7% and 39.5% of the total cellular DiO@Zr-PL-HDL and DiO@Zr-AI-HDL in tumors, respectively. CONCLUSION: We have developed (89)Zr-labeled TAM imaging agents based on the natural nanoparticle rHDL. In an orthotopic mouse model of breast cancer, we have demonstrated their specificity for macrophages, a result that was corroborated by flow cytometry. Quantitative macrophage PET imaging with our (89)Zr-rHDL imaging agents could be valuable for noninvasive monitoring of TAM immunology and targeted treatment.

In mouse models of atherosclerosis, normalization of hyperlipidemia promotes macrophage emigration and regression of atherosclerotic plaques in part by the Liver X Receptor (LXR)-mediated induction of the chemokine receptor CCR7. Here we report that LXRalpha serine 198 (S198) phosphorylation modulates CCR7 expression. Low levels of S198 phosphorylation are observed in plaque macrophages in the regression environment where high levels of CCR7 expression are observed. Consistent with these findings, CCR7 gene expression in human and mouse macrophages cell lines is induced when LXRalpha at S198 is non-phosphorylated. In bone marrow derived-macrophages (BMDMs) we also observe induction of CCR7 by ligands that promote non-phosphorylated LXRalpha S198 and this is lost in LXR deficient BMDMs. LXRalpha occupancy at the CCR7 promoter is enhanced and histone modifications associated with gene repression are reduced in RAW264.7 cells expressing non-phosphorylated (RAW-LXRalphaS198A) compared to phosphorylated LXRalpha (RAW-LXRalphaWT). Expression profiling from ligand treated RAW-LXRalphaS198A compared to RAW-LXRalphaWT cells revealed induction of cell migratory and anti-inflammatory genes, and repression of pro-inflammatory genes. Modeling of LXRalpha S198 in non-phosphorylated and phosphorylated states identified phosphorylation-dependent conformational changes in the hinge region commensurate with sites for protein interaction. Therefore, gene transcription is regulated by LXRalpha S198 phosphorylation including anti-atherogenic genes like CCR7.

Inflammation drives atherosclerotic plaque progression and rupture, and is a compelling therapeutic target. Consequently, attenuating inflammation by reducing local macrophage accumulation is an appealing approach. This can potentially be accomplished by either blocking blood monocyte recruitment to the plaque or increasing macrophage apoptosis and emigration. Because macrophage proliferation was recently shown to dominate macrophage accumulation in advanced plaques, locally inhibiting macrophage proliferation may reduce plaque inflammation and produce long-term therapeutic benefits. To test this hypothesis, we used nanoparticle-based delivery of simvastatin to inhibit plaque macrophage proliferation in apolipoprotein E deficient mice (Apoe-/- ) with advanced atherosclerotic plaques. This resulted in rapid reduction of plaque inflammation and favorable phenotype remodeling. We then combined this short-term nanoparticle intervention with an eight-week oral statin treatment, and this regimen rapidly reduced and continuously suppressed plaque inflammation. Our results demonstrate that pharmacologically inhibiting local macrophage proliferation can effectively treat inflammation in atherosclerosis.

High-density lipoprotein (HDL) is a natural nanoparticle that exhibits an intrinsic affinity for atherosclerotic plaque macrophages. Its natural targeting capability as well as the option to incorporate lipophilic payloads, e.g., imaging or therapeutic components, in both the hydrophobic core and the phospholipid corona make the HDL platform an attractive nanocarrier. To realize controlled release properties, we developed a hybrid polymer/HDL nanoparticle composed of a lipid/apolipoprotein coating that encapsulates a poly(lactic-co-glycolic acid) (PLGA) core. This novel HDL-like nanoparticle (PLGA-HDL) displayed natural HDL characteristics, including preferential uptake by macrophages and a good cholesterol efflux capacity, combined with a typical PLGA nanoparticle slow release profile. In vivo studies carried out with an ApoE knockout mouse model of atherosclerosis showed clear accumulation of PLGA-HDL nanoparticles in atherosclerotic plaques, which colocalized with plaque macrophages. This biomimetic platform integrates the targeting capacity of HDL biomimetic nanoparticles with the characteristic versatility of PLGA-based nanocarriers.

OBJECTIVE: We previously showed that cholesterol loading in vitro converts mouse aortic vascular smooth muscle cells (VSMC) from a contractile state to one resembling macrophages. In human and mouse atherosclerotic plaques, it has become appreciated that approximately 40% of cells classified as macrophages by histological markers may be of VSMC origin. Therefore, we sought to gain insight into the molecular regulation of this clinically relevant process. APPROACH AND RESULTS: VSMC of mouse (or human) origin were incubated with cyclodextrin-cholesterol complexes for 72 hours, at which time the expression at the protein and mRNA levels of contractile-related proteins was reduced and of macrophage markers increased. Concurrent was downregulation of miR-143/145, which positively regulate the master VSMC differentiation transcription factor myocardin. Mechanisms were further probed in mouse VSMC. Maintaining the expression of myocardin or miR-143/145 prevented and reversed phenotypic changes caused by cholesterol loading. Reversal was also seen when cholesterol efflux was stimulated after loading. Notably, despite expression of macrophage markers, bioinformatic analyses showed that cholesterol-loaded cells remained closer to the VSMC state, consistent with impairment in classical macrophage functions of phagocytosis and efferocytosis. In apoE-deficient atherosclerotic plaques, cells positive for VSMC and macrophage markers were found lining the cholesterol-rich necrotic core. CONCLUSIONS: Cholesterol loading of VSMC converts them to a macrophage-appearing state by downregulating the miR-143/145-myocardin axis. Although these cells would be classified by immunohistochemistry as macrophages in human and mouse plaques, their transcriptome and functional properties imply that their contributions to atherogenesis would not be those of classical macrophages.

Liver X receptor (LXR) signaling pathways regulate lipid metabolism and inflammation, which has generated widespread interest in developing synthetic LXR agonists as potential therapeutics for the management of atherosclerosis. In this study, it is demonstrated that nanoparticles (NPs) containing the synthetic LXR agonist GW3965 (NP-LXR) exert anti-inflammatory effects and inhibit the development of atherosclerosis without causing hepatic steatosis. These NPs are engineered through self-assembly of a biodegradable diblock poly(lactide-co-glycolide)-b-poly(ethylene glycol) (PLGA-b-PEG) copolymer. NP-LXR is significantly more effective than free GW3965 at inducing LXR-target gene expression and suppressing inflammatory factors in macrophages in vitro and in vivo. Additionally, the NPs elicit negligible lipogenic gene stimulation in the liver. Using the Ldlr (-/-) mouse model of atherosclerosis, abundant colocalization of fluorescently labeled NPs within plaque macrophages following systemic administration is seen. Notably, six intravenous injections of NP-LXR over 2 weeks markedly reduce the CD68-positive cell (macrophage) content of plaques (by 50%) without increasing total cholesterol or triglycerides in the liver and plasma. Together, these findings identify GW3965-encapsulated PLGA-b-PEG NPs as a promising nanotherapeutic approach to combat atherosclerosis, providing the benefits of LXR agonists without their adverse effects on hepatic and plasma lipid metabolism.

OBJECTIVE: Diabetic hypertriglyceridemia is thought to be primarily driven by increased hepatic de novo lipogenesis. However, experiments in animal models indicated that insulin deficiency should decrease hepatic de novo lipogenesis and reduce plasma triglyceride levels. APPROACH AND RESULTS: To address the discrepancy between human data and genetically altered mouse models, we investigated whether insulin-deficient diabetic mice had triglyceride changes that resemble those in diabetic humans. Streptozotocin-induced insulin deficiency increased plasma triglyceride levels in mice. Contrary to the mouse models with impaired hepatic insulin receptor signaling, insulin deficiency did not reduce hepatic triglyceride secretion and de novo lipogenesis-related gene expression. Diabetic mice had a marked decrease in postprandial triglycerides clearance, which was associated with decreased lipoprotein lipase and peroxisome proliferator-activated receptor alpha mRNA levels in peripheral tissues and decreased lipoprotein lipase activity in skeletal muscle, heart, and brown adipose tissue. Diabetic heterozygous lipoprotein lipase knockout mice had markedly elevated fasting plasma triglyceride levels and prolonged postprandial triglycerides clearance. CONCLUSIONS: Insulin deficiency causes hypertriglyceridemia by decreasing peripheral lipolysis and not by an increase in hepatic triglycerides production and secretion.

We tested whether a high fat diet (HFD) containing the inflammatory dietary fatty acid palmitate or insulin deficient diabetes altered the remodeling of atherosclerotic plaques in LDL receptor knockout (Ldlr-/-) mice. Cholesterol reduction was achieved by using a helper-dependent adenovirus (HDAd) carrying the gene for the low-density lipoprotein receptor (Ldlr; HDAd-LDLR). After injection of the HDAd-LDLR, mice consuming either HFD, which led to insulin resistance but not hyperglycemia, or low fat diet (LFD), showed regression compared to baseline. However there was no difference between the two groups in terms of atherosclerotic lesion size, or CD68+ cell and lipid content. Because of the lack of effects of these two diets, we then tested whether viral-mediated cholesterol reduction would lead to defective regression in mice with greater hyperglycemia. In both normoglycemic and streptozotocin (STZ)-treated hyperglycemic mice, HDAd-LDLR significantly reduced plasma cholesterol levels, decreased atherosclerotic lesion size, reduced macrophage area and lipid content, and increased collagen content of plaque in the aortic sinus. However, reductions in anti-inflammatory and ER stress-related genes were less pronounced in STZ-diabetic mice compared to non-diabetic mice. In conclusion, HDAd-mediated Ldlr gene therapy is an effective and simple method to induce atherosclerosis regression in Ldlr-/- mice in different metabolic states.