Insights from Murine Studies on the Site Specificity of Atherosclerosis.

Atherosclerosis is an inflammatory reaction that develops at specific regions within the artery wall and at specific sites of the arterial tree over a varying time frame in response to a variety of risk factors. The mechanisms that account for the interaction of systemic factors and atherosclerosis-susceptible regions of the arterial tree to mediate this site-specific development of atherosclerosis are not clear. The dynamics of blood flow has a major influence on where in the arterial tree atherosclerosis develops, priming the site for interactions with atherosclerotic risk factors and inducing cellular and molecular participants in atherogenesis. But how this accounts for lesion development at various locations along the vascular tree across differing time frames still requires additional study. Currently, murine models are favored for the experimental study of atherogenesis and provide the most insight into the mechanisms that may contribute to the development of atherosclerosis. Based largely on these studies, in this review, we discuss the role of hemodynamic shear stress, SR-B1, and other factors that may contribute to the site-specific development of atherosclerosis.

Pig and Mouse Models of Hyperlipidemia and Atherosclerosis.

Atherosclerosis is a chronic inflammatory disorder that is the underlying cause of most cardiovascular disease. Resident cells of the artery wall and cells of the immune system participate in atherogenesis. This process is influenced by plasma lipoproteins, genetics, and the hemodynamics of the blood flow in the artery. A variety of animal models have been used to study the pathophysiology and mechanisms that contribute to atherosclerotic lesion formation. No model is ideal as each has its own advantages and limitations with respect to manipulation of the atherogenic process and modeling human atherosclerosis and lipoprotein profile. In this chapter we will discuss pig and mouse models of experimental atherosclerosis. The similarity of pig lipoprotein metabolism and the pathophysiology of the lesions in these animals with that of humans is a major advantage. While a few genetically engineered pig models have been generated, the ease of genetic manipulation in mice and the relatively short time frame for the development of atherosclerosis has made them the most extensively used model. Newer approaches to induce hypercholesterolemia in mice have been developed that do not require germline modifications. These approaches will facilitate studies on atherogenic mechanisms.

A lysosome-targeted DNA nanodevice selectively targets macrophages to attenuate tumours.

Activating CD8+ T cells by antigen cross-presentation is remarkably effective at eliminating tumours. Although this function is traditionally attributed to dendritic cells, tumour-associated macrophages (TAMs) can also cross-present antigens. TAMs are the most abundant tumour-infiltrating leukocyte. Yet, TAMs have not been leveraged to activate CD8+ T cells because mechanisms that modulate their ability to cross-present antigens are incompletely understood. Here we show that TAMs harbour hyperactive cysteine protease activity in their lysosomes, which impedes antigen cross-presentation, thereby preventing CD8+ T cell activation. We developed a DNA nanodevice (E64-DNA) that targets the lysosomes of TAMs in mice. E64-DNA inhibits the population of cysteine proteases that is present specifically inside the lysosomes of TAMs, improves their ability to cross-present antigens and attenuates tumour growth via CD8+ T cells. When combined with cyclophosphamide, E64-DNA showed sustained tumour regression in a triple-negative-breast-cancer model. Our studies demonstrate that DNA nanodevices can be targeted with organelle-level precision to reprogram macrophages and achieve immunomodulation in vivo.

Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis.

Cancer cell genetic variability and similarity to host cells have stymied development of broad anti-cancer therapeutics. Our innate immune system evolved to clear genetically diverse pathogens and limit host toxicity; however, whether/how innate immunity can produce similar effects in cancer is unknown. Here, we show that human, but not murine, neutrophils release catalytically active neutrophil elastase (ELANE) to kill many cancer cell types while sparing non-cancer cells. ELANE proteolytically liberates the CD95 death domain, which interacts with histone H1 isoforms to selectively eradicate cancer cells. ELANE attenuates primary tumor growth and produces a CD8+T cell-mediated abscopal effect to attack distant metastases. Porcine pancreatic elastase (ELANE homolog) resists tumor-derived protease inhibitors and exhibits markedly improved therapeutic efficacy. Altogether, our studies suggest that ELANE kills genetically diverse cancer cells with minimal toxicity to non-cancer cells, raising the possibility of developing it as a broad anti-cancer therapy.

Atherosclerosis: cell biology and lipoproteins.

PURPOSE OF REVIEW: Lipoproteins have significant role in both the promotion and prevention of atherosclerosis. This brief review will focus on recent reports on relationship between HDL and HDL subclasses and their composition and function, the role of apoC-III in metabolism of triglyceride-rich lipoproteins, the impact of Lipoprotein (a) (Lp(a)) on endothelial cells, and the mechanism of uptake of aggregated LDL by macrophages. RECENT FINDINGS: The complexity of the protein and lipid content of murine and human HDL and their relationship to its cholesterol efflux capacity have been examined. HDL has also been shown to have both antiatherogenic and proatherogenic properties. The relationship between apoC-III and LPL activity, apoprotein E mediated clearance of triglyceride-rich lipoproteins and the potential importance of apoC-III in the increased risk of cardiovascular disease in type 1 diabetics has been investigated. Oxidized phospholipid in Lp(a) promotes endothelial cells inflammatory and glycolytic responses. TLR4 participates in the uptake of aggregated LDL to contribute to foam cell formation. SUMMARY: These studies contribute to our mechanistic understanding of how lipoproteins contribute to atherogenesis and identify potential therapeutic targets.

CD1d Selectively Down Regulates the Expression of the Oxidized Phospholipid-Specific E06 IgM Natural Antibody in Ldlr-/- Mice.

Natural antibodies (NAbs) are important regulators of tissue homeostasis and inflammation and are thought to have diverse protective roles in a variety of pathological states. E06 is a T15 idiotype IgM NAb exclusively produced by B-1 cells, which recognizes the phosphocholine (PC) head group in oxidized phospholipids on the surface of apoptotic cells and in oxidized LDL (OxLDL), and the PC present on the cell wall of Streptococcus pneumoniae. Here we report that titers of the E06 NAb are selectively increased several-fold in Cd1d-deficient mice, whereas total IgM and IgM antibodies recognizing other oxidation specific epitopes such as in malondialdehyde-modified LDL (MDA-LDL) and OxLDL were not increased. The high titers of E06 in Cd1d-deficient mice are not due to a global increase in IgM-secreting B-1 cells, but they are specifically due to an expansion of E06-secreting splenic B-1 cells. Thus, CD1d-mediated regulation appeared to be suppressive in nature and specific for E06 IgM-secreting cells. The CD1d-mediated regulation of the E06 NAb generation is a novel mechanism that regulates the production of this specific oxidation epitope recognizing NAb.

Invariant Natural Killer T-Cells and Total CD1d Restricted Cells Differentially Influence Lipid Metabolism and Atherosclerosis in Low Density Receptor Deficient Mice.

Natural killer T (NKT) cells are a distinct subset of lymphocytes that bridge the innate and adaptive immune response and can be divided into type I invariant NKT cells (iNKT) and type II NKT cells. The objective of this study is to examine the effects of NKT cell on lipid metabolism and the initiation and progression of atherosclerosis in LDL receptor deficient (LDLR-/-) mice. Mice were fed an atherogenic diet for 4 or 8 weeks and plasma lipids, lipoproteins, and atherosclerosis were measured. The selective absence of iNKT cells in Jα18-/-LDLR-/- mice led to an increase in plasma cholesterol levels in female mice. Transgenic Vα14tg/LDLR-/- mice with elevated numbers of iNKT cells had increased late atherosclerosis of the innominate artery, though absence of either iNKT cells or all NKT cells and other CD1d expressing cells had varying effects on atherosclerotic lesion burden in the ascending aortic arch and aortic root. These studies not only highlight the potential modulatory role played by NKT cells in atherosclerosis and lipid metabolism, but also raise the possibility that divergent roles may be played by iNKT and CD1d restricted cells such as type II NKT cells or other CD1d expressing cells.

Apoproteins E, A-I, and SAA in Macrophage Pathobiology Related to Atherogenesis.

Macrophages are core cellular elements of both early and advanced atherosclerosis. They take up modified lipoproteins and become lipid-loaded foam cells and secrete factors that influence other cell types in the artery wall involved in atherogenesis. Apoproteins E, AI, and SAA are all found on HDL which can enter the artery wall. In addition, apoE is synthesized by macrophages. These three apoproteins can promote cholesterol efflux from lipid-loaded macrophages and have other functions that modulate macrophage biology. Mimetic peptides based on the sequence or structure of these apoproteins replicate some of these properties and are potential therapeutic agents for the treatment of atherosclerosis to reduce cardiovascular diseases.

Apoprotein E and Reverse Cholesterol Transport.

Apoprotein E (apoE) is a multifunctional protein. Its best-characterized function is as a ligand for low-density lipoprotein (LDL) receptor family members to mediate the clearance of apoB-containing atherogenic lipoproteins. Among its other functions, apoE is involved in cholesterol efflux, especially from cholesterol-loaded macrophage foam cells and other atherosclerosis-relevant cells, and in reverse cholesterol transport. Reverse cholesterol transport is a mechanism by which excess cellular cholesterol is transported via lipoproteins in the plasma to the liver where it can be excreted from the body in the feces. This process is thought to have a role in the attenuation of atherosclerosis. This review summarizes studies on the role of apoE in cellular cholesterol efflux and reverse cholesterol transport and discusses the identification of apoE mimetic peptides that may promote these pathways.

Obesity and Insulin Resistance Promote Atherosclerosis through an IFNγ-Regulated Macrophage Protein Network.

Type 2 diabetes (T2D) is associated with increased risk for atherosclerosis; however, the mechanisms underlying this relationship are poorly understood. Macrophages, which are activated in T2D and causatively linked to atherogenesis, are an attractive mechanistic link. Here, we use proteomics to show that diet-induced obesity and insulin resistance (obesity/IR) modulate a pro-atherogenic "macrophage-sterol-responsive-network" (MSRN), which, in turn, predisposes macrophages to cholesterol accumulation. We identify IFNγ as the mediator of obesity/IR-induced MSRN dysregulation and increased macrophage cholesterol accumulation and show that obesity/IR primes T cells to increase IFNγ production. Accordingly, myeloid cell-specific deletion of the IFNγ receptor (Ifngr1-/-) restores MSRN proteins, attenuates macrophage cholesterol accumulation and atherogenesis, and uncouples the strong relationship between hyperinsulinemia and aortic root lesion size in hypercholesterolemic Ldlr-/- mice with obesity/IR, but does not affect these parameters in Ldlr-/- mice without obesity/IR. Collectively, our findings identify an IFNγ-macrophage pathway as a mechanistic link between obesity/IR and accelerated atherogenesis.

Small Intestine Microbiota Regulate Host Digestive and Absorptive Adaptive Responses to Dietary Lipids.

The gut microbiota play important roles in lipid metabolism and absorption. However, the contribution of the small bowel microbiota of mammals to these diet-microbe interactions remains unclear. We determine that germ-free (GF) mice are resistant to diet-induced obesity and malabsorb fat with specifically impaired lipid digestion and absorption within the small intestine. Small bowel microbes are essential for host adaptation to dietary lipid changes by regulating gut epithelial processes involved in their digestion and absorption. In addition, GF mice conventionalized with high-fat diet-induced jejunal microbiota exhibit increased lipid absorption even when fed a low-fat diet. Conditioned media from specific bacterial strains directly upregulate lipid absorption genes in murine proximal small intestinal epithelial organoids. These findings indicate that proximal gut microbiota play key roles in host adaptability to dietary lipid variations through mechanisms involving both the digestive and absorptive phases and that these functions may contribute to conditions of over- and undernutrition.

Hypercholesterolemia-Induced Loss of Flow-Induced Vasodilation and Lesion Formation in Apolipoprotein E-Deficient Mice Critically Depend on Inwardly Rectifying K+ Channels.

BACKGROUND: Hypercholesterolemia-induced decreased availability of nitric oxide (NO) is a major factor in cardiovascular disease. We previously established that cholesterol suppresses endothelial inwardly rectifying K+ (Kir) channels and that Kir2.1 is an upstream mediator of flow-induced NO production. Therefore, we tested the hypothesis that suppression of Kir2.1 is responsible for hypercholesterolemia-induced inhibition of flow-induced NO production and flow-induced vasodilation (FIV). We also tested the role of Kir2.1 in the development of atherosclerotic lesions. METHODS AND RESULTS: Kir2.1 currents are significantly suppressed in microvascular endothelial cells exposed to acetylated-low-density lipoprotein or isolated from apolipoprotein E-deficient (Apoe-/- ) mice and rescued by cholesterol depletion. Genetic deficiency of Kir2.1 on the background of hypercholesterolemic Apoe-/- mice, Kir2.1+/-/Apoe-/- exhibit the same blunted FIV and flow-induced NO response as Apoe-/- or Kir2.1+/- alone, but while FIV in Apoe-/- mice can be rescued by cholesterol depletion, in Kir2.1+/-/Apoe-/- mice cholesterol depletion has no effect on FIV. Endothelial-specific overexpression of Kir2.1 in arteries from Apoe-/- and Kir2.1+/-/Apoe-/- mice results in full rescue of FIV and NO production in Apoe-/- mice with and without the addition of a high-fat diet. Conversely, endothelial-specific expression of dominant-negative Kir2.1 results in the opposite effect. Kir2.1+/-/Apoe-/- mice also show increased lesion formation, particularly in the atheroresistant area of descending aorta. CONCLUSIONS: We conclude that hypercholesterolemia-induced reduction in FIV is largely attributable to cholesterol suppression of Kir2.1 function via the loss of flow-induced NO production, whereas the stages downstream of flow-induced Kir2.1 activation appear to be mostly intact. Kir2.1 channels also have an atheroprotective role.

Deficiency of Invariant Natural Killer T Cells Does Not Protect Against Obesity but Exacerbates Atherosclerosis in Ldlr-/- Mice.

Obesity is a chronic inflammatory state characterized by altered levels of adipose tissue immune cell populations. Natural killer T (NKT) cells are CD1d restricted lymphocyte subsets that recognize lipid antigens whose level decreases in obese adipose tissue. However, studies in mice with deficiency or increased levels of NKT cells have yielded contradictory results, so the exact role of these cells in obesity and adipose tissue inflammation is not yet established. We previously showed that Ldlr-/- mice with excess invariant NKT (iNKT) cells demonstrate significant weight gain, adiposity, metabolic abnormalities, and atherosclerosis. The current study evaluates the effects of NKT cell deficiency on obesity, associated metabolic changes, and atherosclerosis in Jα18-/-Ldlr-/- (lacking iNKT cells) and Cd1d-/-Ldlr-/- (lacking invariant and type II NKT cells) mice, and control mice were fed an obesogenic diet (high fat, sucrose, cholesterol) for 16 weeks. Contrary to expectations, Ja18-/-Ldlr-/- mice gained significantly more weight than Ldlr-/- or Cd1d-/-Ldlr-/- mice, developed hypertriglyceridemia, and had worsened adipose tissue inflammation. All the mice developed insulin resistance and hepatic triglyceride accumulation. Ja18-/-Ldlr-/- mice also had increased atherosclerotic lesion area. Our findings suggest that iNKT cells exacerbates the metabolic, inflammatory, and atherosclerotic features of diet-induced obesity. Further work is required to unravel the paradox of an apparently similar effect of iNKT cell surplus and depletion on obesity.

T Cells in Atherosclerosis in Ldlr-/- and Apoe-/- Mice.

Atherosclerosis is the underlying basis for most cardiovascular diseases. It is a chronic inflammation affecting the arterial intima and is promoted by hypercholesterolemia. Cells of both the innate and adaptive immune systems contribute to this inflammation with macrophages and T cells being the most abundant immune cells in the atherosclerotic plaques. In this review, we discuss the studies that examined the role of T cells and T cell subsets in Apoe-/- and Ldlr-/- murine models of atherosclerosis. While there is a general consensus that Th1 cells are pro-atherogenic and regulatory T cells are atheroprotective, the role of other subsets is more ambiguous. In addition, the results in the two models of atherosclerosis do not always yield similar results. Additional studies in the two murine models using cell specific gene manipulations are needed.