Endotoxinemia Accelerates Atherosclerosis Through Electrostatic Charge-Mediated Monocyte Adhesion.

BACKGROUND: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here, we use a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation Methods: Acute infection was mimicked by injection of a single dose of lipopolysaccharide into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy. RESULTS: Lipopolysaccharide treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional myeloid cell accumulation. Lipopolysaccharide treatment led to the deposition of NETs along the arterial lumen, and inhibition of NET release annulled lesion expansion during endotoxinemia, thus suggesting that NETs regulate myeloid cell recruitment. To study the mechanism of monocyte adhesion to NETs, we used in vitro adhesion assays and biophysical approaches. In these experiments, NET-resident histone H2a attracted monocytes in a receptor-independent, surface charge-dependent fashion. Therapeutic neutralization of histone H2a by antibodies or by in silico designed cyclic peptides enables us to reduce luminal monocyte adhesion and lesion expansion during endotoxinemia. CONCLUSIONS: Our study shows that NET-associated histone H2a mediates charge-dependent monocyte adhesion to NETs and accelerates atherosclerosis during endotoxinemia.

Pro-Angiogenic Macrophage Phenotype to Promote Myocardial Repair.

BACKGROUND: Heart failure following myocardial infarction (MI) remains one of the major causes of death worldwide, and its treatment is a crucial challenge of cardiovascular medicine. An attractive therapeutic strategy is to stimulate endogenous mechanisms of myocardial regeneration. OBJECTIVES: This study evaluates the potential therapeutic treatment with annexin A1 (AnxA1) to induce cardiac repair after MI. METHODS: AnxA1 knockout (AnxA1-/-) and wild-type mice underwent MI induced by ligation of the left anterior descending coronary artery. Cardiac functionality was assessed by longitudinal echocardiographic measurements. Histological, fluorescence-activated cell sorting, dot blot analysis, and in vitro/ex vivo studies were used to assess the myocardial neovascularization, macrophage content, and activity in response to AnxA1. RESULTS: AnxA1-/- mice showed a reduced cardiac functionality and an expansion of proinflammatory macrophages in the ischemic area. Cardiac macrophages from AnxA1-/- mice exhibited a dramatically reduced ability to release the proangiogenic mediator vascular endothelial growth factor (VEGF)-A. However, AnxA1 treatment enhanced VEGF-A release from cardiac macrophages, and its delivery in vivo markedly improved cardiac performance. The positive effect of AnxA1 treatment on cardiac performance was abolished in wild-type mice transplanted with bone marrow derived from Cx3cr1creERT2Vegfflox/flox or in mice depleted of macrophages. Similarly, cardioprotective effects of AnxA1 were obtained in pigs in which full-length AnxA1 was overexpressed by use of a cardiotropic adeno-associated virus. CONCLUSIONS: AnxA1 has a direct action on cardiac macrophage polarization toward a pro-angiogenic, reparative phenotype. AnxA1 stimulated cardiac macrophages to release high amounts of VEGF-A, thus inducing neovascularization and cardiac repair.

Organ-Specific Mechanisms of Transendothelial Neutrophil Migration in the Lung, Liver, Kidney, and Aorta.

Immune responses are dependent on the recruitment of leukocytes to the site of inflammation. The classical leukocyte recruitment cascade, consisting of capture, rolling, arrest, adhesion, crawling, and transendothelial migration, is thoroughly studied but mostly in model systems, such as the cremasteric microcirculation. This cascade paradigm, which is widely accepted, might be applicable to many tissues, however recruitment mechanisms might substantially vary in different organs. Over the last decade, several studies shed light on organ-specific mechanisms of leukocyte recruitment. An improved awareness of this matter opens new therapeutic windows and allows targeting inflammation in a tissue-specific manner. The aim of this review is to summarize the current understanding of the leukocyte recruitment in general and how this varies in different organs. In particular we focus on neutrophils, as these are the first circulating leukocytes to reach the site of inflammation. Specifically, the recruitment mechanism in large arteries, as well as vessels in the lungs, liver, and kidney will be addressed.

Chrono-pharmacological Targeting of the CCL2-CCR2 Axis Ameliorates Atherosclerosis.

Onset of cardiovascular complications as a consequence of atherosclerosis exhibits a circadian incidence with a peak in the morning hours. Although development of atherosclerosis extends for long periods of time through arterial leukocyte recruitment, we hypothesized that discrete diurnal invasion of the arterial wall could sustain atherogenic growth. Here, we show that myeloid cell recruitment to atherosclerotic lesions oscillates with a peak during the transition from the activity to the resting phase. This diurnal phenotype is regulated by rhythmic release of myeloid cell-derived CCL2, and blockade of its signaling abolished oscillatory leukocyte adhesion. In contrast, we show that myeloid cell adhesion to microvascular beds peaks during the early activity phase. Consequently, timed pharmacological CCR2 neutralization during the activity phase caused inhibition of atherosclerosis without disturbing microvascular recruitment. These findings demonstrate that chronic inflammation of large vessels feeds on rhythmic myeloid cell recruitment, and lay the foundation for chrono-pharmacology-based therapy.

Human Neutrophil Peptide 1 Limits Hypercholesterolemia-induced Atherosclerosis by Increasing Hepatic LDL Clearance.

Increases in plasma LDL-cholesterol have unequivocally been established as a causal risk factor for atherosclerosis. Hence, strategies for lowering of LDL-cholesterol may have immediate therapeutic relevance. Here we study the role of human neutrophil peptide 1 (HNP1) in a mouse model of atherosclerosis and identify its potent atheroprotective effect both upon transgenic overexpression and therapeutic delivery. The effect was found to be due to a reduction of plasma LDL-cholesterol. Mechanistically, HNP1 binds to apolipoproteins enriched in LDL. This interaction facilitates clearance of LDL particles in the liver via LDL receptor. Thus, we here identify a non-redundant mechanism by which HNP1 allows for reduction of LDL-cholesterol, a process that may be therapeutically instructed to lower cardiovascular risk.

Protective Aptitude of Annexin A1 in Arterial Neointima Formation in Atherosclerosis-Prone Mice-Brief Report.

OBJECTIVE: Restenosis as a consequence of arterial injury is aggravated by inflammatory pathways. Here, we investigate the role of the proresolving protein annexin A1 (AnxA1) in healing after wire injury. APPROACH AND RESULTS: Apoe-/- and Apoe-/-Anxa1-/- mice were subjected to wire injury while fed a high-cholesterol diet. Subsequently, localization of AnxA1 and AnxA1 plasma levels were examined. AnxA1 was found to localize within endothelial cells and macrophages in the neointima. Levels of AnxA1 in the plasma and its lesional expression negatively correlated with neointima size, and in the absence of AnxA1, neointima formation was aggravated by the accumulation and proliferation of macrophages. In contrast, reendothelialization and smooth muscle cell infiltration were not affected in Apoe-/-Anxa1-/- mice. CONCLUSIONS: AnxA1 is protective in healing after wire injury and could, therefore, be an attractive therapeutic compound to prevent from restenosis after vascular damage.

Resolving Lipid Mediators Maresin 1 and Resolvin D2 Prevent Atheroprogression in Mice.

RATIONALE: Atheroprogression is a consequence of nonresolved inflammation, and currently a comprehensive overview of the mechanisms preventing resolution is missing. However, in acute inflammation, resolution is known to be orchestrated by a switch from inflammatory to resolving lipid mediators. Therefore, we hypothesized that lesional lipid mediator imbalance favors atheroprogression. OBJECTIVE: To understand the lipid mediator balance during atheroprogression and to establish an interventional strategy based on the delivery of resolving lipid mediators. METHODS AND RESULTS: Aortic lipid mediator profiling of aortas from Apoe-/- mice fed a high-fat diet for 4 weeks, 8 weeks, or 4 months revealed an expansion of inflammatory lipid mediators, Leukotriene B4 and Prostaglandin E2, and a concomitant decrease of resolving lipid mediators, Resolvin D2 (RvD2) and Maresin 1 (MaR1), during advanced atherosclerosis. Functionally, aortic Leukotriene B4 and Prostaglandin E2 levels correlated with traits of plaque instability, whereas RvD2 and MaR1 levels correlated with the signs of plaque stability. In a therapeutic context, repetitive RvD2 and MaR1 delivery prevented atheroprogression as characterized by halted expansion of the necrotic core and accumulation of macrophages along with increased fibrous cap thickness and smooth muscle cell numbers. Mechanistically, RvD2 and MaR1 induced a shift in macrophage profile toward a reparative phenotype, which secondarily stimulated collagen synthesis in smooth muscle cells. CONCLUSIONS: We present evidence for the imbalance between inflammatory and resolving lipid mediators during atheroprogression. Delivery of RvD2 and MaR1 successfully prevented atheroprogression, suggesting that resolving lipid mediators potentially represent an innovative strategy to resolve arterial inflammation.

Annexin A1 counteracts chemokine-induced arterial myeloid cell recruitment.

RATIONALE: Chemokine-controlled arterial leukocyte recruitment is a crucial process in atherosclerosis. Formyl peptide receptor 2 (FPR2) is a chemoattractant receptor that recognizes proinflammatory and proresolving ligands. The contribution of FPR2 and its proresolving ligand annexin A1 to atherosclerotic lesion formation is largely undefined. OBJECTIVE: Because of the ambivalence of FPR2 ligands, we here investigate the role of FPR2 and its resolving ligand annexin A1 in atherogenesis. METHODS AND RESULTS: Deletion of FPR2 or its ligand annexin A1 enhances atherosclerotic lesion formation, arterial myeloid cell adhesion, and recruitment. Mechanistically, we identify annexin A1 as an endogenous inhibitor of integrin activation evoked by the chemokines CCL5, CCL2, and CXCL1. Specifically, the annexin A1 fragment Ac2-26 counteracts conformational activation and clustering of integrins on myeloid cells evoked by CCL5, CCL2, and CXCL1 through inhibiting activation of the small GTPase Rap1. In vivo administration of Ac2-26 largely diminishes arterial recruitment of myeloid cells in a FPR2-dependent fashion. This effect is also observed in the presence of selective antagonists to CCR5, CCR2, or CXCR2, whereas Ac2-26 was without effect when all 3 chemokine receptors were antagonized simultaneously. Finally, repeated treatment with Ac2-26 reduces atherosclerotic lesion sizes and lesional macrophage accumulation. CONCLUSIONS: Instructing the annexin A1-FPR2 axis harbors a novel approach to target arterial leukocyte recruitment. With the ability of Ac2-26 to counteract integrin activation exerted by various chemokines, delivery of Ac2-26 may be superior in inhibition of arterial leukocyte recruitment when compared with blocking individual chemokine receptors.

Repeatable, Inducible Micro-RNA-Based Technology Tightly Controls Liver Transgene Expression.

Inducible systems for gene expression emerge as a new class of artificial vectors offering temporal and spatial exogenous control of gene expression. However, most inducible systems are less efficient in vivo and lack the target-organ specificity. In the present study, we have developed and optimized an oligonucleotide-based inducible system for the in vivo control of transgenes in the liver. We generated a set of simple, inducible plasmid-vectors based on the addition of four units of liver-specific miR-122 target sites to the 3'untranslated region of the gene of interest. Once the vector was delivered into hepatocytes this modification induced a dramatic reduction of gene expression that could be restored by the infusion of an antagomir for miR-122. The efficiency of the system was tested in vivo, and displayed low background and strong increase in gene expression upon induction. Moreover, gene expression was repeatedly induced even several months after the first induction showing no toxic effect in vivo. By combining tissue-specific control elements with antagomir treatment we generated, optimized and validated a robust inducible system that could be used successfully for in vivo experimental models requiring tight and cyclic control of gene expression.

Nanomedicine-based strategies for treatment of atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the arterial wall that arises from an imbalanced lipid metabolism and a maladaptive inflammatory response. Despite intensive research on mechanisms underlying atherosclerotic lesion formation and progression during the past decade, translation of this knowledge into the clinic is scarce. Although developments have primarily been made in the area of antitumor therapy, recent advances have shown the potential of nanomedicine-based treatment strategies for atherosclerosis. Here we describe the features of currently available nanomedical formulations that have been optimized for atherosclerosis treatment, and we further describe how they can be instructed to target inflammatory processes in the arterial wall. Despite their limitations, nanomedical applications might hold promise for personalized medicine, and further efforts are needed to improve atherosclerosis-specific targeting.

Gene therapy for advanced melanoma: selective targeting and therapeutic nucleic acids.

Despite recent advances, the treatment of malignant melanoma still results in the relapse of the disease, and second line treatment mostly fails due to the occurrence of resistance. A wide range of mutations are known to prevent effective treatment with chemotherapeutic drugs. Hence, approaches with biopharmaceuticals including proteins, like antibodies or cytokines, are applied. As an alternative, regimens with therapeutically active nucleic acids offer the possibility for highly selective cancer treatment whilst avoiding unwanted and toxic side effects. This paper gives a brief introduction into the mechanism of this devastating disease, discusses the shortcoming of current therapy approaches, and pinpoints anchor points which could be harnessed for therapeutic intervention with nucleic acids. We bring the delivery of nucleic acid nanopharmaceutics into perspective as a novel antimelanoma therapeutic approach and discuss the possibilities for melanoma specific targeting. The latest reports on preclinical and already clinical application of nucleic acids in melanoma are discussed.

Systemic TNFα gene therapy synergizes with liposomal doxorubicine in the treatment of metastatic cancer.

Tumor necrosis factor alpha (TNFα) is a potent antitumoral cytokine, either killing tumor cells directly or affecting the tumor vasculature leading to enhanced accumulation of macromolecular drugs. Due to dose limiting side effects systemic administration of TNFα protein at therapeutically active doses is precluded. With gene vectors, tumor restricted TNFα expression can be achieved and in principle synergize with chemotherapy. Synthetic gene carriers based on polyamines were intravenously injected, which either passively accumulate within the tumor or specifically target the epidermal growth factor receptor. A single intravenous injection of TNFα gene vector promoted accumulation of liposomal doxorubicine (Doxil) in murine neuroblastoma and human hepatoma by enhancing tumor endothelium permeability. The expression of transgenic TNFα was restricted to tumor tissue. Three treatment cycles with TNFα gene vectors and Doxil significantly delayed tumor growth in subcutaneous murine Neuro2A neuroblastoma. Also tumors re-growing after initial treatment were successfully treated in a fourth cycle pointing at the absence of resistance mechanisms. Systemic Neuro2A metastases or human LS174T colon carcinoma metastases in liver were also successfully treated with this combined approach. In conclusion, this schedule opens the possibility for the efficient treatment of tumors metastases otherwise not accessible for macromolecular drug carriers.

Diaminopropionic acid lipopeptides: characterization studies of polyplexes aimed at pDNA delivery.

Here we report a novel class of peptides-d-diaminopropionic acids (Dap)-for gene delivery. These peptides have attractive properties for gene delivery, and the advantage that they can be easily manipulated in relation to their composition, abiding with tailored-design. We characterized the toxicological and biophysical properties of DNA particles resulting from the interaction of the nucleic acid with a series of Dap(8) peptides conjugated to different alkyl groups. These peptides formed small and homogenous DNA particle populations that protected against DNase I degradation at non-toxic concentrations. However, despite the similarity between these peptides and others that are arginine-rich, and efficient vectors, functional studies suggest the need for additional modifications in the carriers to improve their DNA delivery efficiency. Taken together, these studies underscore the relevance of the overall structure of the carrier and the complexity of designing from scratch a carrier.

PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation.

Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.

A peptide-based vector for efficient gene transfer in vitro and in vivo.

Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo.

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.

Non-viral nanovectors for gene delivery: factors that govern successful therapeutics.

IMPORTANCE OF THE FIELD: Gene therapy is regarded as one of the most promising therapeutic approaches, as it has the potential to treat disorders by correcting malformations at the nucleic acids. AREAS COVERED IN THIS REVIEW: Some of the most recent developments in the process of plasmid DNA vector design and formulation are reviewed with a special focus on: different formulations of nanovectors and a summary of successful cases reported; requirements for systemic administration; and functionalization of the nanocarriers by use of targeting entities. WHAT THE READER WILL GAIN: An understanding of the different physiological barriers and a comprehensive review of the recent strategies used to overcome these obstacles. Particular attention is given to formulations for intravenous administration, colloidal stability properties and different targeting entities used. TAKE HOME MESSAGE: Overall, vector formulation must take into account the administration route and inherent physiological barriers. Critical parameters for the success of pDNA nanovectors are: particles size, colloidal stability of the formulation and interaction between the carrier and plasmid DNA. Highly relevant is the fact that this interaction should be balanced to offer protection to degradation as well as allow dissociation of the therapeutic nucleic acid for obtaining maximal activity.

Temperature-assisted cyclic hybridization (TACH): an improved method for supercoiled DNA hybridization.

Accurate hybridization is dependent on the ratio between sequence-specific and unspecific binding. Dissociation of unspecifically bound, while maintaining specifically hybridized, nucleic acids are key steps to obtain a well-defined complex. We have developed a new method, temperature-assisted, cyclic hybridization (TACH), which increases cognate binding at the expense of unspecific hybridization. The method was used for optimizing binding of peptide nucleic acid (PNA) to supercoiled plasmids and has several advantages over previous methods: (1) it reduces the required amount of bis-PNA by three- to fourfold; (2) it results in less unspecific binding; (3) it extends cooperative hybridization, from 3 bp to 5 bp between two adjacent binding sites; and (4) it decreases the aggregation of bis-PNA. This method might be extended to other forms of hybridizations including the use of additional nucleic acids analogs, such as locked nucleic acid (LNA) and, also, to other areas where PNAs are used such as fluorescence in situ hybridization (FISH), microarrays, or in vivo plasmid delivery.