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Summary: Current pharmacologic treatments for atherosclerosis do not completely protect patients; additional protection can be achieved by dietary modifications, such as a low-cholesterol/low-fat diet (LCLFD), that mediate plaque stabilization and inflammation reduction. However, this lifestyle modification can be challenging for patients. Unfortunately, incomplete understanding of the underlying mechanisms has thwarted efforts to mimic the protective effects of a LCLFD. Here, we report that the tricarboxylic acid cycle intermediate itaconate (ITA), produced by plaque macrophages, is key to diet-induced plaque resolution. ITA is produced by immunoresponsive gene 1 (IRG1), which we observe is highly elevated in myeloid cells of vulnerable plaques and absent from early or stable plaques in mice and humans. We additionally report development of an ITA-conjugated lipid nanoparticle that accumulates in plaque and bone marrow myeloid cells, epigenetically reduces inflammation via H3K27ac deacetylation, and reproduces the therapeutic effects of LCLFD-induced plaque resolution in multiple atherosclerosis models.

Abstract Cardiac muscle targeting is a notoriously difficult task. Although various nanoparticle (NP) and adeno-associated viral (AAV) strategies with heart tissue tropism have been developed, their performance remains suboptimal. Significant off-target accumulation of i.v.-delivered pharmacotherapies has thwarted development of disease-modifying cardiac treatments, such as gene transfer and gene editing, that may address both rare and highly prevalent cardiomyopathies and their complications. Here, we present an intriguing discovery: cargo-less, safe poly (lactic-co-glycolic acid) particles that drastically improve heart delivery of AAVs and NPs. Our lead formulation is referred to as ePL (enhancer polymer). We show that ePL increases selectivity of AAVs and virus-like NPs (VLNPs) to the heart and de-targets them from the liver. Serotypes known to have high (AAVrh.74) and low (AAV1) heart tissue tropisms were tested with and without ePL. We demonstrate up to an order of magnitude increase in heart-to-liver accumulation ratios in ePL-injected mice. We also show that ePL exhibits AAV/NP-independent mechanisms of action, increasing glucose uptake in the heart, increasing cardiac protein glycosylation, reducing AAV neutralizing antibodies, and delaying blood clearance of AAV/NPs. Current approaches utilizing AAVs or NPs are fraught with challenges related to the low transduction of cardiomyocytes and life-threatening immune responses; our study introduces an exciting possibility to direct these modalities to the heart at reduced i.v. doses and, thus, has an unprecedented impact on drug delivery and gene therapy. Based on our current data, the ePL system is potentially compatible with any therapeutic modality, opening a possibility of cardiac targeting with numerous pharmacological approaches. Graphical Abstract

Abstract Background It has been well recognized that toxicity of fine ambient air particulate matter (PM2.5) may depend on its chemical constituents, including components such as soluble metals that may theoretically exert distinctive effects. We have recently demonstrated an important effect of PM2.5 on metabolic function. Since transition metals, such as nickel (Ni), represent an important component of exposure in certain environments, and may significantly influence the toxicity of inhalational exposure, we investigated the effects of Ni as a variable component of ambient PM2.5 exposure. Methods Male ApoE knockout mice were exposed to filtered air (FA), fine-sized nickel sulfate particles alone (Ni) at 0.44 μg/m3, concentrated ambient air PM2.5 (CAPs) at a mean of 70 μg/m3, or CAPs+Ni in Tuxedo, NY, 6 hours/day, 5 days/week, for 3 months. Results Exposure to Ni, irrespective of co-exposure to CAPs, resulted in body weight gain, while exposure to CAPs+Ni significantly enhanced fasting glucose and worsened insulin resistance measures (HOMA-IR), when compared with exposure to CAPs alone. CAPs+Ni exposure induced a significant decrease in phosphorylation of AMP-activated protein kinase (AMPK) α. Exposure to Ni or CAPs+Ni significantly induced microcirculatory dysfunction and increased monocytic cell infiltration into lung and adipose, and decreased uncoupling protein 1 expression at gene and protein levels and several brown adipocyte-specific genes in adipose tissue. Conclusions Ni exposure has effects on metabolic and inflammatory parameters that are comparable to that of CAPs. Additionally, Ni synergistically exacerbates CAPs-induced adverse effects on some of, but not all of, these parameters, that may be mediated via the AMPK signaling pathway. These findings have important implications for inhaled transition metal toxicity that may exert synergistic effects with other PM2.5 components.

Native nanostructured lipoproteins such as low- and high-density lipoproteins (LDL and HDL) are powerful tools for the targeted delivery of drugs and imaging agents. While the cellular recognition of well-known HDL-based carriers occurs via interactions with an HDL receptor, the selective delivery and uptake of LDL particles by target cells are more complex. The most well-known mode of LDL-based delivery is via the interaction between apolipoprotein B (Apo-B) – the main protein of LDL – and the low-density lipoprotein receptor (LDLR). LDLR is expressed in the liver, adipocytes, and macrophages, and thus selectively delivers LDL carriers to these cells and tissues. Moreover, the elevated expression of LDLR in tumor cells indicates a role for LDL in the targeted delivery of chemotherapy drugs. In addition, chronic inflammation associated with hypercholesterolemia (i.e., high levels of endogenous LDL) can be abated by LDL carriers, which outcompete the deleterious oxidized LDL for uptake by macrophages. In this case, synthetic LDL nanocarriers act as ‘eat-me’ signals and exploit mechanisms of native LDL uptake for targeted drug delivery and imaging. Lastly, recent studies have shown that the delivery of LDL-based nanocarriers to macrophages via fluid-phase pinocytosis is a promising tool for atherosclerosis imaging. Hence, the present review summarizes the use of natural and synthetic LDL-based carriers for drug delivery and imaging and discusses various mechanisms of targeting.

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BACKGROUND The resolution of inflammation is an active phenomenon important for switching off inflammatory processes once the harmful stimuli are removed and facilitate the return to homeostasis. Specialized pro-resolving mediators (SPMs), such as lipoxin A4, resolvin D1, and resolvin E1, derived from ω-3 or ω-6 polyunsaturated fatty acids, are crucial for the resolution of inflammation. We hypothesized that SPMs are decreased in hypertension which contributes to the acetylcholine-induced contraction in resistance arteries, which are well known to be mediated by leukotrienes and prostaglandins. Moreover, treatment with SPMs will decrease this contraction via formyl peptide receptor-2 (FPR-2) in resistance arteries from spontaneously hypertensive rats (SHR). METHODS AND RESULTS We performed a comprehensive eicosanoid lipid panel analysis, and our data showed for the first time that precursors of SPMs are decreased in SHR, limiting the production of SPMs and resolution of inflammation in vivo. This phenomenon was associated with an increase in lipid peroxidation in resistance arteries. Although SPMs did not abolish acetylcholine-induced contraction, these lipid mediators improved endothelial function in arteries from SHR via FPR-2 activation at nanomolar concentrations. SPMs also buffered TNF-α-induced reactive oxygen species generation in endothelial cells from C57Bl/6 mice. CONCLUSIONS We suggest that FPR-2 and SPMs could be revealed as a new target or therapeutic agent to improve vascular function in arteries from hypertensive rats. [ABSTRACT FROM AUTHOR]

Although statin therapy is a cornerstone of current low density lipoprotein (LDL)-lowering strategies, there is a need for additional therapies to incrementally lower plasma LDL cholesterol. In this study, we investigated the effect of several methylenedioxyphenol derivatives in regulating LDL cholesterol through induction of LDL receptor (LDLR). INV-403, a modified methylenedioxyphenol derivative, increased LDLR mRNA and protein expression in HepG2 cells in a dose- and time-dependent fashion. These effects were apparent even under conditions of HMG-CoA reductase inhibition. Electrophoresis migration shift assays demonstrated that INV-403 activates SREBP2 but not SREBP1c, with immunoblot analysis showing an increased expression of the mature form of SREBP2. Knockdown of SREBP2 reduced the effect of INV-403 on LDLR expression. The activation of SREBP2 by INV-403 is partly mediated by Akt/GSK3β pathways through inhibition of phosphorylation-dependent degradation by ubiquitin-proteosome pathway. Treatment of C57Bl/6j mice with INV-403 for two weeks increased hepatic SREBP2 levels (mature form) and upregulated LDLR with concomitant lowering of plasma LDL levels. Transient expression of a LDLR promoter-reporter construct, a SRE-mutant LDLR promoter construct, and a SRE-only construct in HepG2 cells revealed an effect predominantly through a SRE-dependent mechanism. INV-403 lowered plasma LDL cholesterol levels through LDLR upregulation. These results indicate a role for small molecule approaches other than statins for lowering LDL cholesterol.

Exteriorized phosphatidylserine (PS) residues in apoptotic cells trigger rapid phagocytosis by macrophage scavenger receptor pathways. Mimicking apoptosis with liposomes containing PS may represent an attractive approach for molecular imaging of atherosclerosis. We investigated the utility of paramagnetic gadolinium liposomes enriched with PS (Gd-PS) in imaging atherosclerotic plaque. Gd-PS-containing Gd-conjugated lipids, fluorescent rhodamine, and PS were prepared and characterized. Cellular uptake in RAW macrophages (fluorescent uptake of rhodamine) was studied on a fluorescence plate reader, while Gd-PS-induced alteration in T1 relaxivity was evaluated using a 1.5 T MRI scanner. RAW cells demonstrate PS-dependent uptake of across a range of concentrations (2, 6, 12, and 20%) in comparison to control liposomes with no PS (0%). In vivo performance of Gd-PS was evaluated in the ApoE−/− mouse model by collection of serial T1 weighted gradient echo MR images using an 11.7 T MRI system and revealed rapid and significant enhancement of the aortic wall that was seen for at least 4 h after injection. Gd-PS-enriched liposomes enhance atherosclerotic plaque and colocalize with macrophages in experimental atherosclerosis.

Inflammation and oxidative stress play fundamental roles in the pathogenesis of atherosclerosis. Myeloperoxidase has been extensively implicated as a key mediator of inflammatory and redox-dependent processes in atherosclerosis. However, the effect of synthetic myeloperoxidase inhibitors on atherosclerosis has been insufficiently studied. In this study, ApoE(-/-) mice were randomized to low- and high-dose INV-315 groups for 16 weeks on high-fat diet. INV-315 resulted in reduced plaque burden and improved endothelial function in response to acetylcholine. These effects occurred without adverse events or changes in body weight or blood pressure. INV-315 treatment resulted in a decrease in iNOS gene expression, superoxide production and nitrotyrosine content in the aorta. Circulating IL-6 and inflammatory CD11b(+)/Ly6G(low)/7/4(hi) monocytes were significantly decreased in response to INV-315 treatment. Acute pretreatment with INV-315 blocked TNFα-mediated leukocyte adhesion in cremasteric venules and inhibited myeloperoxidase activity. Cholesterol efflux was significantly increased by high-dose INV-315 via ex-vivo reverse cholesterol transport assays. Our results suggest that myeloperoxidase inhibition may exert anti-atherosclerotic effects via inhibition of oxidative stress and enhancement of cholesterol efflux. These findings demonstrate a role for pharmacologic modulation of myeloperoxidase in atherosclerosis.