Jessica L. Feig

The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques

Atherosclerotic plaque formation is fueled by the persistence of lipid-laden macrophages in the artery wall. The mechanisms by which these cells become trapped, thereby establishing chronic inflammation, remain unknown. Here we found that netrin-1, a neuroimmune guidance cue, was secreted by macrophages in human and mouse atheroma, where it inactivated the migration of macrophages toward chemokines linked to their egress from plaques. Acting via its receptor, UNC5b, netrin-1 inhibited the migration of macrophages directed by the chemokines CCL2 and CCL19, activation of the actin-remodeling GTPase Rac1 and actin polymerization. Targeted deletion of netrin-1 in macrophages resulted in much less atherosclerosis in mice deficient in the receptor for low-density lipoprotein and promoted the emigration of macrophages from plaques. Thus, netrin-1 promoted atherosclerosis by retaining macrophages in the artery wall. Our results establish a causative role for negative regulators of leukocyte migration in chronic inflammation.

Hypoxia Induces Netrin-1 and Unc5b in Atherosclerotic Plaques Mechanism for Macrophage Retention and Survival

Objective— Hypoxia is intimately linked to atherosclerosis and has become recognized as a primary impetus of inflammation. We recently demonstrated that the neuroimmune guidance cue netrin-1 ( Ntn1 ) inhibits macrophage emigration from atherosclerotic plaques, thereby fostering chronic inflammation. However, the mechanisms governing netrin-1 expression in atherosclerosis are not well understood. In this study, we investigate the role of hypoxia in regulating expression of netrin-1 and its receptor uncoordinated-5-B receptor (Unc5b) in plaque macrophages and its functional consequences on these immune cells. Approach and Results— We show by immunostaining that netrin-1 and Unc5b are expressed in macrophages in hypoxia-rich regions of human and mouse plaques. In vitro , Ntn1 and Unc5b mRNA are upregulated in macrophages treated with oxidized low-density lipoprotein or inducers of oxidative stress (CoCl2, dimethyloxalylglycine, 1% O2). These responses are abrogated by inhibiting hypoxia-inducible transcription factor (HIF)-1α, indicating a causal role for this transcription factor in regulating Ntn1 and Unc5b expression in macrophages. Indeed, using promoter-luciferase reporter genes, we show that Ntn1 - and Unc5b -promoter activities are induced by oxidized low-density lipoprotein and require HIF-1α. Correspondingly, J774 macrophages overexpressing active HIF-1α show increased netrin-1 and Unc5b expression and reduced migratory capacity compared with control cells, which was restored by blocking the effects of netrin-1. Finally, we show that netrin-1 protects macrophages from apoptosis under hypoxic conditions in a HIF-1α–dependent manner. Conclusions— These findings provide a molecular mechanism by which netrin-1 and its receptor Unc5b are expressed in atherosclerotic plaques and implicate hypoxia and HIF-1α–induced netrin-1/Unc5b in sustaining inflammation by inhibiting the emigration and promoting the survival of lesional macrophages. # Significance {#article-title-39}Objective—Hypoxia is intimately linked to atherosclerosis and has become recognized as a primary impetus of inflammation. We recently demonstrated that the neuroimmune guidance cue netrin-1 (Ntn1) inhibits macrophage emigration from atherosclerotic plaques, thereby fostering chronic inflammation. However, the mechanisms governing netrin-1 expression in atherosclerosis are not well understood. In this study, we investigate the role of hypoxia in regulating expression of netrin-1 and its receptor uncoordinated-5-B receptor (Unc5b) in plaque macrophages and its functional consequences on these immune cells. Approach and Results—We show by immunostaining that netrin-1 and Unc5b are expressed in macrophages in hypoxia-rich regions of human and mouse plaques. In vitro, Ntn1 and Unc5b mRNA are upregulated in macrophages treated with oxidized low-density lipoprotein or inducers of oxidative stress (CoCl2, dimethyloxalylglycine, 1% O2). These responses are abrogated by inhibiting hypoxia-inducible transcription factor (HIF)-1&agr;, indicating a causal role for this transcription factor in regulating Ntn1 and Unc5b expression in macrophages. Indeed, using promoter-luciferase reporter genes, we show that Ntn1- and Unc5b-promoter activities are induced by oxidized low-density lipoprotein and require HIF-1&agr;. Correspondingly, J774 macrophages overexpressing active HIF-1&agr; show increased netrin-1 and Unc5b expression and reduced migratory capacity compared with control cells, which was restored by blocking the effects of netrin-1. Finally, we show that netrin-1 protects macrophages from apoptosis under hypoxic conditions in a HIF-1&agr;–dependent manner. Conclusions—These findings provide a molecular mechanism by which netrin-1 and its receptor Unc5b are expressed in atherosclerotic plaques and implicate hypoxia and HIF-1&agr;–induced netrin-1/Unc5b in sustaining inflammation by inhibiting the emigration and promoting the survival of lesional macrophages.

Adenosine A2A Receptor and TNF-α Regulate the Circadian Machinery of the Human Monocytic THP-1 Cells

Morning stiffness and increased symptoms of inflammatory arthritis are among the most common manifestations of rheumatoid arthritis (RA). Tumor necrosis alpha (TNF-α), an important mediator of inflammation in RA, regulates the circadian expression of clock proteins, and adenosine A2A receptors (A2AR) mediate many of the anti-inflammatory and antirheumatic actions of methotrexate, the cornerstone drug in the treatment of RA. We found that A2AR activation and TNF-α activated the clock core loop of the human monocytic THP-1 cell line. We further observed that interleukin (IL)-10, but not IL-12, mRNA expression fluctuates in a circadian fashion and that TNF-α and A2AR stimulation combined increased IL-10 expression. Interestingly, TNF-α, but not CGS21680, dramatically inhibited IL-12 mRNA expression. The demonstration that A2AR and TNF-α regulate the intrinsic circadian clock in immune cells provides an explanation for both the pathologic changes in circadian rhythms in RA and for the adverse circadian effects of methotrexate, such as fatigue.

Macrophages, dendritic cells, and regression of atherosclerosis.

Atherosclerosis is the number one cause of death in the Western world. It results from the interaction between modified lipoproteins and cells such as macrophages, dendritic cells (DCs), T cells, and other cellular elements present in the arterial wall. This inflammatory process can ultimately lead to the development of complex lesions, or plaques, that protrude into the arterial lumen. Ultimately, plaque rupture and thrombosis can occur leading to the clinical complications of myocardial infarction or stroke. Although each of the cell types plays roles in the pathogenesis of atherosclerosis, the focus of this review will be primarily on the macrophages and DCs. The role of these two cell types in atherosclerosis is discussed, with a particular emphasis on their involvement in atherosclerosis regression.

Statins, atherosclerosis regression and HDL: Insights from within the plaque

The idea that atheroma can regress is no longer a dream. We and others have discovered that decreasing the lipid content can directly lead to macrophage egress and plaque healing. The question, however, has remained as to how to translate these findings to the bedside. Taking advantage of imaging modalities such as intravascular ultrasound (IVUS) and near infrared spectroscopy (NIRS), we demonstrated in the YELLOW (Reduction in Yellow Plaque by Intensive Lipid Lowering Therapy) trial that short term treatment of high dose rosuvastatin treatment can lead to a decrease in lipid content in plaques. It is important to note that optical coherence tomography (OCT), a high resolution imaging modality, was not performed during the first study and therefore, only a very limited assessment of the effect of statin therapy on measures of plaque stabilization could be made. The YELLOW II trial is the first to our knowledge to determine whether these data can be extrapolated and how it relates to HDL function, alterations in macrophage gene expression, and plaque morphology. While tremendous progress has been made, our research serves as a reminder that angiography is simply luminography and it is features such as thin cap fibroatheroma and lipid burden, for example, that likely modulate the syndromes seen in clinical practice. Ongoing studies such as ours may provide novel pathways for diagnosis and therapy, with the ultimate goal of reducing the burden of cardiovascular disease.

How MicroRNAs Modify Protein Production

OVERVIEW It is generally accepted that the flow of genetic information from genomic DNA to functional protein is not unidirectional and linear. Rather, numerous molecular pathways regulate the processes of replication, transcription, and translation to fine-tune the output of protein-coding genes. Specifically, RNA interference is a biological process in which specialized small RNAs—short interfering RNAs (siRNAs) or microRNAs (miRNAs)—posttranscriptionally regulate the expression of specific target genes. In the December 2013 Research Techniques Made Simple, Nambudiri and Widlund described siRNAs and applications in investigative dermatology (Nambudiri and Widlund, 2013). Here, we outline how miRNAs modulate expression of genes relevant to skin pathology by inhibiting protein translation.

The antiviral drug tenofovir, an inhibitor of Pannexin-1-mediated ATP release, prevents liver and skin fibrosis by downregulating adenosine levels in the liver and skin.

Background Fibrosing diseases are a leading cause of morbidity and mortality worldwide and, therefore, there is a need for safe and effective antifibrotic therapies. Adenosine, generated extracellularly by the dephosphorylation of adenine nucleotides, ligates specific receptors which play a critical role in development of hepatic and dermal fibrosis. Results of recent clinical trials indicate that tenofovir, a widely used antiviral agent, reverses hepatic fibrosis/cirrhosis in patients with chronic hepatitis B infection. Belonging to the class of acyclic nucleoside phosphonates, tenofovir is an analogue of AMP. We tested the hypothesis that tenofovir has direct antifibrotic effects in vivo by interfering with adenosine pathways of fibrosis using two distinct models of adenosine and A2AR-mediated fibrosis. Methods Thioacetamide (100mg/kg IP)-treated mice were treated with vehicle, or tenofovir (75mg/kg, SubQ) (n = 5–10). Bleomycin (0.25U, SubQ)-treated mice were treated with vehicle or tenofovir (75mg/kg, IP) (n = 5–10). Adenosine levels were determined by HPLC, and ATP release was quantitated as luciferase-dependent bioluminescence. Skin breaking strength was analysed and H&E and picrosirus red-stained slides were imaged. Pannexin-1expression was knocked down following retroviral-mediated expression of of Pannexin-1-specific or scrambled siRNA. Results Treatment of mice with tenofovir diminished adenosine release from the skin of bleomycin-treated mice and the liver of thioacetamide-treated mice, models of diffuse skin fibrosis and hepatic cirrhosis, respectively. More importantly, tenofovir treatment diminished skin and liver fibrosis in these models. Tenofovir diminished extracellular adenosine concentrations by inhibiting, in a dose-dependent fashion, cellular ATP release but not in cells lacking Pannexin-1. Conclusions These studies suggest that tenofovir, a widely used antiviral agent, could be useful in the treatment of fibrosing diseases.

The role of HDL in plaque stabilization and regression: basic mechanisms and clinical implications.

On the basis of studies that extend back to the early 1900s, regression and stabilization of atherosclerosis in humans has progressed from being a concept to one that is achievable. Successful attempts at regression generally applied robust measures to improve plasma lipoprotein profiles. Possible mechanisms responsible for lesion shrinkage include decreased retention of atherogenic apolipoprotein B within the arterial wall, efflux of cholesterol and other toxic lipids from plaques, emigration of lesional foam cells out of the arterial wall, and influx of healthy phagocytes that remove necrotic debris as well as other components of the plaque. Currently available clinical agents, however, still fail to stop most cardiovascular events. For years, HDL has been considered the ‘good cholesterol.’ Clinical intervention studies to causally link plasma HDL-C levels to decreased progression or to the regression of atherosclerotic plaques are relatively few because of the lack of therapeutic agents that can selectively and potently increase HDL-C. The negative results of studies that were carried out have led to uncertainty as to the role that HDL plays in atherosclerosis. It is becoming clearer, however, that HDL function rather than quantity is most crucial and, therefore, discovery of agents that enhance the quality of HDL should be the goal.

The Regression of Atherosclerosis: The Power of Multimodality Imaging

Coronary artery disease remains a leading cause of death in the Western world. It is well known that the reduction of cholesterol levels by statin therapy is associated with significant decreases in plaque burden. However, a critical question has been the ability of statin therapy to lead to lipid egress from plaque and subsequent plaque stabilization. This is crucial since lipid-rich coronary plaques are at increased risk for rupture and thrombus leading to events. We recently addressed this issue by conducting the YELLOW (reduction in yellow plaque by aggressive lipid-lowering therapy) trial. We reported that short-term intensive statin therapy reduces lipid content as assessed by near infrared spectroscopy (NIRS) in obstructive lesions. Histopathological studies suggest that the majority of acute coronary events are related to occlusive thrombus formation after disruption of a thin-cap fibroatheroma (TCFA) overlying a large necrotic lipid core. Optical coherence tomography (OCT) is an imaging modality with powerful resolution to allow for plaque characterization including the identification of TCFA. Yet, it still remains unknown as to the efficacy of high dose statin therapy for enhancing anatomic features of plaque stabilization. The YELLOW II Trial was therefore designed with the goal of combining the utilization of OCT and NIRS in the coronary arteries to extend our initial findings as well as link them to changes in plaque morphology with alterations in lipoprotein biology, HDL function and macrophage behaviour.

Sirolimus Stents, Positive Remodeling, Late Incomplete Stent Apposition, Oh My!

A 52 year old female with a history of hyperlipidemia and hypothyroidism presented to the emergency department with intermittent diffuse chest discomfort and throat tightness after walking approximately one block. Her past medical history is significant for two mid left anterior descending artery (mLAD) Cypher sirolimus stents (2.5 mm × 23 mm, 2.5 mm × 8 mm) placed in 2008 when she presented with similar symptoms. She has no allergies or past surgical history. Family history is notable for a mother with hypertension and a father with diabetes. She drinks socially and has never smoked or uses other substances. Her medications were aspirin, amlodipine, pitavastatin, synthroid and a multivitamin. Of note, plavix was discontinued one year after stent placement. Electrocardiogram demonstrated normal sinus rhythm without any significant ST changes. Physical exam was unremarkable. Cardiac enzymes were negative. During stress echocardiogram, the patient was able to complete 11 METS without any symptoms. There was no inducible ischemia or wall motion abnormalities at >85% MPHR (mean peak heart rate) with a rest ejection fraction of 60%. CT angiography (CTA) revealed two sequential stents in which in stent restenosis (ISR) was unable to be excluded.