Original Article Aortic Iron Overload With Oxidative Stress and Inflammation in Human and Murine Abdominal Aortic Aneurysm Hisashi Sawada,* Hiroyuki Hao,* Yoshiro Naito, Makiko Oboshi, Shinichi Hirotani, Masataka Mitsuno, Yuji Miyamoto, Seiichi Hirota, Tohru Masuyama Objective—Although iron is an essential element for maintaining physiological function, excess iron leads to tissue damage caused by oxidative stress and inflammation. Oxidative stress and inflammation play critical roles for the development of abdominal aortic aneurysm (AAA). However, it has not been investigated whether iron plays a role in AAA formation through oxidative stress and inflammation. We, therefore, examined whether iron is involved in the pathophysiology of AAA formation using human AAA walls and murine AAA models. Approach and Results—Human aortic walls were collected from 53 patients who underwent cardiovascular surgery (nonAAA=34; AAA=19). Murine AAA was induced by infusion of angiotensin II to apolipoprotein E knockout mice. Iron was accumulated in human and murine AAA walls compared with non-AAA walls. Immunohistochemistry showed that both 8-hydroxy-2′-deoxyguanosine and CD68-positive areas were increased in AAA walls compared with non-AAA walls. The extent of iron accumulated area positively correlated with that of 8-hydroxy-2′-deoxyguanosine expression area and macrophage infiltration area in human and murine AAA walls. We next investigated the effects of dietary iron restriction on AAA formation in mice. Iron restriction reduced the incidence of AAA formation with attenuation of oxidative stress and inflammation. Aortic expression of transferrin receptor 1, intracellular iron transport protein, was increased in human and murine AAA walls, and transferrin receptor 1–positive area was similar to areas where iron accumulated and F4/80 were positive. Conclusions—Iron is involved in the pathophysiology of AAA formation with oxidative stress and inflammation. Dietary iron restriction could be a new therapeutic strategy for AAA progression.   (Arterioscler Thromb Vasc Biol. 2015;35:00-00. DOI: 10.1161/ATVBAHA.115.305586.) Key Words: aortic aneurysm, abdominal ◼ inflammation ◼ iron ◼ oxidative stress

A

bdominal aortic aneurysm (AAA) is a common vascular disease, which occurs in ≈4% to 8% of men aged 65 to 80 years.1 Aortic rupture is the most feared clinical consequence of AAA progression, resulting in mortality in ≈90% of cases.2–4 Because AAA usually progresses without symptoms, AAA is often discovered in advanced stage. At present, surgical aortic replacement and endovascular stent graft repair are performed as standard definitive therapies for AAA. However, there is no effective medical therapy to prevent aortic rupture in AAA. Although several studies have reported the pathophysiology of AAA, the mechanisms of AAA formation are largely unknown. Therefore, it is necessary to investigate the molecular pathophysiology of AAA to find noninvasive strategies for the prevention of AAA. Iron is an essential element for maintaining physiological function. However, excess iron causes tissue damage

by oxidative stress via the Fenton/Haber–Weiss reaction.5 Therefore, iron is involved in the pathophysiology of several diseases including cardiovascular diseases. In fact, we have previously shown that iron accumulation and superoxide production are observed in the renal tubules of a rat model of chronic kidney disease.6 Lee et al7 have shown that iron accumulation is observed in the atherosclerotic lesions of apolipoprotein E knockout mice. Most recently, Martinez-Pinna et al8 have demonstrated that iron is deposited in human AAA walls. Meanwhile, we have also reported that dietary iron restriction (IR) prevents the development of hypertension and proteinuria with inhibition of oxidative stress and inflammation in Dahl salt-sensitive hypertensive rats.9 Although oxidative stress and inflammation are well known to be involved in the development of AAA formation, it has not been investigated whether iron is associated with the pathophysiology of AAA through

Received on: November 1, 2014; final version accepted on: March 30, 2015. From the Cardiovascular Division, Department of Internal Medicine (H.S., Y.N., M.O., S. Hirotani, T.M.), Department of Surgical Pathology (H.H., S. Hirota), and Department of Cardiovascular Surgery (M.M., Y.M.), Hyogo College of Medicine, Nishinomiya, Hyogo, Japan. *These authors contributed equally to this article. The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.115.305586/-/DC1. Correspondence to Yoshiro Naito, MD, PhD, Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo, Japan. E-mail [email protected] © 2015 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org

1

DOI: 10.1161/ATVBAHA.115.305586

2   Arterioscler Thromb Vasc Biol   June 2015

Materials and Methods Materials and Methods are available in the online-only Data Supplement.

Results Iron Accumulation in Human AAA Walls We first examined iron accumulation in human AAA walls. The patient characteristics are shown in the Table. There was no significant difference between 2 groups except the proportions of male and smoking patients. Berlin blue staining revealed that iron was markedly accumulated in AAA walls but was not detected in non-AAA walls (Figure 1A). Aortic iron content of tissue homogenate was also increased in AAA walls compared with non-AAA walls (Figure 1B). Next, we performed immunohistochemistry for 8-hydroxy-2′-deoxyguanosine (8-OHdG) to assess oxidative stress in human aortic walls. Focal 8-OHdG– positive areas were constantly detected in AAA walls, whereas few positive areas were found in non-AAA walls (Figure 1A; Table. 

Patient Characteristics Non-AAA

n Age, y Male/female

AAA

P Value

34

19



74±9

71±11

0.29

B

C 1000 800 600 400 200 0

*

non-AAA

AAA

8-OHdG positive area (%)

Berlin blue positive area (%)

0.8

8-OHdG positive area (%)

15

CD68 positive area (%)

oxidative stress and inflammation. Furthermore, the effects of dietary IR on AAA formation remain unknown. In the present study, we investigated whether iron is involved in the pathophysiology of AAA. In addition, we examined the effects of dietary IR on the development of AAA through oxidative stress and inflammation in a murine model of AAA.

CD68

8-OHdG

Berlin blue

8-hydroxy-2′-deoxyguanosine abdominal aortic aneurysm angiotensin II extracellular signal–regulated kinase iron restriction c-Jun N-terminal kinase matrix metalloproteinase transferrin receptor 1

Aortic iron content (µg/dry g)

8-OHdG AAA AngII ERK IR JNK MMP TfR1

AAA

non-AAA

4

r=0.75 p=0.0003

15 10 5 0

0

1 2 3 Berlin blue positive area (%)

*

0.6 0.4 0.2 0

non-AAA

AAA

*

10 5 0

non-AAA

AAA

*

3 2 1 0

D CD68 positive area (%)

A

Nonstandard Abbreviations and Acronyms

10 8 6 4 2 0

non-AAA

AAA r=0.70 p=0.001

0 1 2 3 Berlin blue positive area (%)

Figure 1. Iron accumulation in human abdominal aortic aneurysm (AAA) walls. A, Representative images of Berlin blue staining, immunohistochemistry for 8-hydroxy-2′-deoxyguanosine (8-OHdG), and CD68 in human non-AAA (n=34) and AAA (n=19) walls. Results of morphometric analysis for these staining are shown in the right column of each staining. B, Aortic iron content in human non-AAA and AAA walls. Correlation between the extent of Berlin blue–positive area and (C) that of 8-OHdG–­ positive area or (D) that of CD68-positive area. *P

Aortic iron overload with oxidative stress and inflammation in human and murine abdominal aortic aneurysm.

Although iron is an essential element for maintaining physiological function, excess iron leads to tissue damage caused by oxidative stress and inflam...
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