Joana R. Viola

Atherosclerosis - A matter of unresolved inflammation.

Atherosclerosis is commonly looked upon as a chronic inflammatory disease of the arterial wall arising from an unbalanced lipid metabolism and a maladaptive inflammatory response. However, atherosclerosis is not merely an inflammation of the vessel wall. In fact, the cardinal signs of unstable atherosclerotic lesions are primarily characteristics of failed resolution of a chronic inflammation. In contrast to acute inflammatory events which are typically self-limiting, atherosclerosis is an unresolved inflammatory condition, lacking the switch from the pro-inflammatory to the pro-resolving phase, the latter characterized by termination of inflammatory cell recruitment, removal of inflammatory cells from the site of inflammation by apoptosis and dead cell clearance, reprogramming of macrophages toward an anti-inflammatory, regenerative phenotype, and finally egress of effector cells and tissue regeneration. Here we present an overview on mechanisms of failed resolution contributing to atheroprogression and deliver a summary of novel therapeutic strategies to restore resolution in inflamed arteries.

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.

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.

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.

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.

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.

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

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.

Double-Strand DNA Sensing Aim2 Inflammasome Regulates Atherosclerotic Plaque Vulnerability

Atherosclerosis, a lipid-driven sterile inflammatory disease of large arteries, and its sequelae such as myocardial infarction and stroke are the most frequent cause of death worldwide. Although atherogenesis is characterized by the accumulation of inflammatory myeloid cells, destabilization of atherosclerotic lesions is dominated by necrotic core expansion and thinning of the fibrous cap. Sterile inflammation is typically induced by host-derived damage-associated molecular patterns that are sensed by pattern recognition receptors. In recent years, cytosolic pattern recognition receptors like the NLRP3 inflammasome have been connected to atherosclerosis, because these can recognize (altered) self-structures such as lesional cholesterol crystals.1 Consequently, the inflammasome complex assembles promoting the release of proinflammatory cytokines interleukin-1β (IL1β) and interleukin-18 (IL18). The recent CANTOS trial (Canakinumab Anti-Inflammatory Thrombosis Outcome Study) lends strong support toward the importance of IL1β in the context of atherosclerosis because therapeutic IL1β neutralization in patients with established atherosclerotic disease revealed a significant reduction in the incidence of a recurrent cardiovascular event.2 Progression of atherosclerosis is characterized by the accumulation of dead cells. Several cell death pathways permitting release of nuclear double-stranded (ds) DNA have been identified including necrosis and release of neutrophil extracellular traps. The amount of extracellular DNA correlates with the risk of cardiovascular events.3 Cytosolic dsDNA can be identified by Absent in melanoma 2 (Aim2) inflammasome leading to the release of IL1β and IL18.4 It is noteworthy that Aim2 has recently been identified in human atherosclerotic lesions in proximity to necrotic cores.5 Here, we study the inflammatory cascade on dsDNA recognition in the context of atherosclerosis by genetically deleting or therapeutically …

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.

Deletion of MFGE8 Inhibits Neointima Formation upon Arterial Damage

1 Institute for Cardiovascular Prevention, LMU Munich, Munich, Germany 2Department of Pathology, Academic Medical Center, Amsterdam University, The Netherlands 3DZHK, Partner Site Munich Heart Alliance, Munich, Germany 4Department of Medicine, Cardiovascular Research Institute, Lung Biology Center, University of California, San Francisco, California, United States 5Department of Physiology and Pharmacology (FyFa), Karolinska Institutet, Stockholm, Sweden 6Department of Medicine, Karolinska Institutet, Stockholm, Sweden