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Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species

Contributing to Insulin Resistance and Atherogenesis

Interview with Dr. Ming, Dr Yang

Basic Sciences, Pharmacology, Genomics and Cardiovascular Pathology

Q: Dr. Ming, Dr Yang, congratulations to your publication which is specially featured in the first issue of JAHA, the new Journal of the American Heart Association. Can you briefly summarize the key findings of the study for us?

A: The key finding of our study is that arginase II promotes inflammatory responses in macrophages in mouse and human cells through mitochondrial dysfunction i.e. increased production of mitochondrial reactive oxygen species. We also demonstrate that deficiency of arginase II gene in the ApoE knockout mice reduces atherosclerotic lesion formation and reveals characteristics of stable plaques. Moreover, arginase II knockout mice show reduced macrophage infiltration in various organs and decreased production of pro-inflammatory cytokines in high‐fat diet–induced obesity and better glucose tolerance and improved insulin sensitivity. The results implicate that targeting arginase II may represent a potential therapeutic strategy in atherosclerosis and in type-II diabetes mellitus.

Q: Two isoforms of arginase exist, however, only the role of arginase I in macrophage inflammatory responses has been extensively studied in the past. Can you speculate why arginase II did not gain more attention in the past? And what was your rationale to focus on this isoform?

A: It is well demonstrated in recent years that macrophages are heterogeneous. In macrophages, iNOS-mediated NO production is widely used as a marker of the pro-inflammatory phenotype (M1) of the cells, while arginase-I is used as a marker of the anti-inflammatory phenotype (M2). This is mainly based on the observations that macrophages which are induced to release more pro-inflammatory cytokines shows markedly increased expression of iNOS, whereas macrophages which release predominantly anti-inflammatory cytokines show up-regulated arginase I levels. It is believed that enhanced expression of arginase I limits the bioavailability of intracellular L-arginine for excessive NO production from iNOS and dampens inflammation and tissue damage, contributing to the anti-inflammatory functions of M2 cells. Arginase I has been thus used as a marker for M2 phenotypic cells. Since arginase I and arginase II have been shown to have the same function in vascular endothelial cells in terms of decreasing eNOS function and arginase I (not arginase II) is consistently shown to be increased in M2 macrophages, this might be the reason that arginase I instead of arginase II is usually analyzed in macrophages and people assume that arginase II should have the same functions as arginase I in the macrophages.
In the past few years, numerous studies including our own demonstrate that arginase II is the major isoform upregulated in the vascular cells from humans and mice contributing to endothelial dysfunction under several pathological conditions including atherosclerosis and diabetes mellitus. Moreover, tissue macrophage infiltration and inflammation play important roles in atherogenesis and obesity-associated insulin resistance or type II diabetes. Yet, little is known about the role of arginase II in macrophage functions. Taking into account that arginase II has been reported to be upregulated in macrophages by pro-inflammatory stimuli such as endotoxin and arginase II expressing macrophages are associated with more advanced atherosclerosis in animal models, we hypothesized that arginase II may promote macrophage inflammatory responses contributing to chronic inflammatory diseases such as atherosclerosis and type-II diabetes.

Q: In the first part of your study, you extensively investigated the role of arginase II in various types of mouse and human macrophages, either cell-line- or bone marrow-derived. Are there differences between these macrophages from different origins? Do human and mouse macrophages behave differentially?

A: Evidences have been presented by literature showing some differences in biological functions between mouse and human macrophages. In support of this notion, we observed upregulation of iNOS levels by endotoxin in mouse macrophages, but not in human monocytic cell lines. They also have biological functions in common. For example, arginase II (not arginase I) expression can be induced in both mouse and human cells upon stimulation by endotoxin. Moreover, downregulation of or deficiency in arginase II reduces production of pro-inflammatory cytokines in both mouse and human cells.

Q: Are the expression levels of the 2 arginase isoforms interdependent, meaning is arginase I downregulated when arginase II is induced? And what happens in knockout mice: is the other arginase isoform upregulated in case of genetic deficiency of one isoform?

A: This is an interesting question. In this study, we did not analyze the inter-regulation of these two arginase isoforms.

Q: What are the underlying mechanisms of pro-inflammatory macrophage responses driven by arginase II?

A: We demonstrate that arginase II promotes pro-inflammatory macrophage responses through amplifying mitochondrial reactive oxygen species production. The anti-oxidant N-acetylcysteine abolishes mitochondrial reactive oxygen species and also the pro-inflammatory responses.   

Q: You then went on to study the in vivo role of arginase II in macrophage inflammatory responses. Which models did you use?

A: Two chronic inflammatory disease mouse models were used. The first model is the high-fat-diet-induced obesity-linked type-II diabetes in arginase II knockout mice, whereby wild type mice serve as control. The second model are the atherosclerosis-prone ApoE knockout mice in which arginase II gene is knocked out, i.e., the ApoE and Arg-II double knockout mice. ApoE knockout mice serve as control.

Q: Is it known whether arginase II is also expressed in other immune or vascular cells? And if so, could this have an impact on the phenotype you observed in your study?

A: Yes. Arginase II is also expressed in endothelial cells and has been shown to play an important role in endothelial dysfunction linking to obesity and atherosclerosis as well as vascular aging as shown by our most recently published study (Yepuri G. et al., Aging Cell. 2012 Aug 29. doi: 10.1111/acel.12001. [Epub ahead of print]). We believe that this could also have an impact on the phenotype we observed in our study. From the therapeutic point of view, targeting arginase II in vivo can improve endothelial function while simultaneously suppressing the pro-inflammatory macrophage responses. This could be beneficial for patients with type-II diabetes, atherosclerosis, and aging-associated cardiovascular dysfunction.

Q: Based on your findings, can we consider arginase II as a marker of the pro-inflammatory M1 macrophage phenotype?

A: We can add arginase II to the list of M1 macrophage makers. However, we think that characterization of M1 phenotype should also include other markers and importantly the functional analysis of macrophages.

Q: To conclude, can you speculate on a clinical perspective? Can we extrapolate your findings or part from it into the clinical practice?

A: As above mentioned, we think that arginase II could be considered as a biomarker for inflammatory diseases. Moreover, development of arginase II specific inhibitors may give rise to a new generation of drugs in treatment of coronary heart disease, type-II diabetes mellitus and associated cardiovascular complications, and vascular aging. 

Q: What are the next steps for the future?

A: We will continue to translate our findings into patients and also wish to develop specific arginase II inhibitors for in vivo pharmacological studies.

Q: Dr. Ming, Dr. Yang, thank you so much for your time and willingness to discuss your exciting findings and share your expertise with us.

A: We thank you and the ESC Working Group Atherosclerosis and Vascular Biology for the interest in our study and for selecting our publication as the interview article of the month!


Xiu-Fen Ming, Angana G. Rajapakse, Gautham Yepuri, Yuyan Xiong, João M. Carvas,Jean Ruffieux, Isabelle Scerri, Zongsong Wu, Katja Popp, Jianhui Li, Claudio Sartori, Urs Scherrer, Brenda R. Kwak, Jean-Pierre Montani and Zhihong Yang
J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.

Notes to editor

Xiu-fen Ming, MD/PhD
Zhihong Yang, MD
Department of Medicine, University of Fribourg - Switzerland

The content of this article reflects the personal opinion of the author/s and is not necessarily the official position of the European Society of Cardiology.

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