The Roles of Peroxiredoxin-2 in Mouse Atherosclerosis Model

Abstract

Reactive oxygen species (ROS), which include superoxide anions and peroxides, induce oxidative stress, contributing to the initiation and progression of cardiovascular diseases involving atherosclerosis. ROS also mediate signaling pathways involved in monocyte adhesion and infiltration, platelet activation, and smooth muscle cell migration. A number of antioxidant enzymes (e.g., superoxide dismutases, catalase, glutathione peroxidases, and peroxiredoxins) regulate ROS in vascular and immune cells. Peroxiredoxins (Prdxs), a family of peroxidases, also play a role in regulating ROS. Prdxs are thiol-specific antioxidant proteins found in mammals, yeast, and bacteria that are classified largely on the basis of having either one or two conserved cysteine residues. Especially, peroxiredoxin-2 (Prdx2) has been reported to regulate pro-inflammatory responses, vascular remodeling, and global oxidative stress. Although Prdx2 has been proposed to retard atherosclerosis development, no direct evidence and mechanisms have been reported. To study the expression pattern of Prdx2 in the vasculature, we stained Prdx2 in the aorta from 8-week-old C57BL/6 and ApoE-/-mice. Prdx2 was not only expressed in the adventitia region of both mice, but was also detected in the endothelial layer of ApoE-/-mice predisposed to atherosclerosis. Next, to test the effect of Prdx2 deficiency on the development of atherosclerosis, we generated double-knockout mice lacking ApoE and Prdx2, and compared plaque formation in Prdx2-/-ApoE-/-and ApoE-/- control littermates at two different stages. At the results, we found Prdx2 deficiency exacerbates atherosclerosis in ApoE-/- mice. Atherosclerosis is initiated by lipids deposited in the subendothelial layer of the artery wall. These lipids and modified LDL damage endothelial cells, inducing the expression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1), and chemotactic molecules including monocyte chemoattractant protein-1 (MCP-1/CCL2). After activating endothelial cells in this way, leukocytes such as monocytes enter the intima by diapedesis and undergo differentiation. To find mechanisms of accelerated plaque formation induced by Prdx2 deficiency, we tested redox-dependent signaling involving nuclear factor-kappa B (NF-κB), activator protein-1 (AP-1), and mitogen-activated protein kinases (MAPKs). Associated with increased atherosclerotic lesions, 20-week-old Prdx2-/-ApoE-/-mice displayed enhanced activation of redox-dependent signaling molecules such as p65, c-Jun, c-Jun N-terminal kinases (JNKs), and p38 MAPK, but not extracellular signal-related kinases (ERKs), compared to the control mice. In addition, the aortic arch of Prdx2-/-ApoE-/-mice showed higher expression of VCAM-1 and ICAM-1, followed by increased transmigration of leukocytes than ApoE-/-control littermates. Increased ROS production has been implicated in hypertension, diabetes mellitus, hypercholesterolemia, restenosis, heart failure, and atherosclerosis. Specifically, overproduction of ROS leads to oxidative stress, which contributes to the pathogenesis of atherosclerosis. To determine whether the increased plaque formation in Prdx2-deficient ApoE-/-mice was due to Prdx2 deficiency in either arterial walls or hematopoietic cells, we conducted bone marrow transplantation experiments. We found that Prdx2 has a major role in inhibiting atherogenic responses in vascular as well as immune cells. Next, we want to examine whether the reduction of elevated endogenous H2O2 levels associated with Prdx2 deficiency could ameliorate atherosclerotic progression. We administered ebselen to Prdx2-/-ApoE-/-mice using osmotic pumps, and found that atherosclerotic plaques in ebselen-treated Prdx2-/-ApoE-/-mice were reduced to a level similar to that in DMSO-treated ApoE-/-mice. Finally, we compared the effect of Prdx2 to other major peroxidases such as glutathione peroxidase 1(GPx1) and catalase on the development of atherosclerosis. We identified Prdx2 and GPx1 were highly expressed compared to catalase in the aortas. In spite of Gpx1 deficiency showing increased tendency to develop atherosclerosis, plaque formation in GPx1-/-ApoE-/-was less than that in Prdx2-/-ApoE-/-mice and catalase deficiency did not affect atherosclerosis development in ApoE-/-mice. In summary, we show that 1) Prdx2 is highly expressed in endothelial and immune cells in atherosclerotic lesions and blocked the increase of endogenous H2O2 by atherogenic stimulation. Deficiency of Prdx2 in apolipoprotein E-deficient (ApoE-/-) mice accelerated plaque formation. 2) Prdx2 deficiency enhanced activation of p65, c-Jun, JNKs and p38 MAPK, and resulted in increased expression of vascular adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1, leading to increased immune-cell adhesion and infiltration into the aortic intima. 3) In bone marrow transplantation experiments, we found that Prdx2 has a major role in inhibiting atherogenic responses in vascular as well as immune cells. Pro-atherogenic effects of Prdx2 deficiency were rescued by administering the antioxidant, ebselen. Compared to deficiency of glutathione peroxidase 1 or catalase, Prdx2 deficiency showed severe predisposition to develop atherosclerosis.

동맥경화증은 고지혈증, 당뇨, 고혈압 등으로 인한 혈관세포의 염증성 자극으로 발병되는 질환으로서 활성산소종(reactive oxygen species, ROS)에 의한 산화적 스트레스가 주요한 원인이라는 사실이 잘 알려져 있다. ROS는 혈관 및 면역세포가 내•외부적인 자극으로 NAD(P)H 산화효소, 잔틴(xanthine) 산화효소, 리폭시게나아제(lipoxygenase)와 같은 효소 시스템을 활성화 시켜 생성된다. ROS는 산화적 스트레스의 생성과 더불어 단핵구 부착 및 침투, 혈소판 활성과 혈관 평활근 세포 이동을 포함한 다양한 병리학적 과정을 유발하는 신호전달에 관여한다. 혈관 및 면역 세포는 ROS를 조절하기 위하여 과산화물 제거효소 (superoxide dismutase, SOD), 카탈라아제(catalase), 글루타티온 과산화효소 (glutathione peroxidase)와 퍼록시레독신(peroxiredoxin)과 같은 많은 항산화 효소를 만들어 낸다. 하지만 ROS생산과 항산화 효소 방어 사이의 국소적 균형이 깨질 때, 동맥경화증과 같은 심혈관 질환이 발생될 수 있다. 퍼록시레독신-2는 황 특이적(thiol-specific) 항산화 효소로 염증반응, 혈관 재형성 및 산화적 스트레스를 조절한다고 보고되었다. 퍼록시레독신-2가 동맥경화증 발병을 줄여줄 수 있을 것이라는 가능성은 제시되었지만 현재까지 직접적인 증거와 작용기작은 밝혀지지 않았다. 이 논문에서 1) 퍼록시레독신 2가 동맥경화 병변의 내피세포와 면역세포에서 활발히 발현하여 동맥경화증을 유발하는 자극에 의해 생성되는 과산화수소를 제거하는 역할을 수행하는 것을 확인하였다. 동맥경화증 모델 마우스인 아포지단백 E 결핍 마우스에서 퍼록시레독신-2가 결핍되었을 때 병변 형성이 증가하는 것을 보였다. 2) 퍼록시레독신-2 결핍은 p65, c-Jun, JNKs와 p38 MAPK의 활성을 증가시키고 그 결과 VCAM-1, ICAM-1 및 MCP-1의 발현을 향상시켜 면역 세포를 혈관벽으로의 부착 및 침착을 증가시킴을 보였다. 3) 골수세포 이식실험을 통해 동맥경화증 발병에서 퍼록시레독신-2의 역할은 면역 세포 및 혈관 구성 세포 모두에서 중요하다는 것을 확인하였고 또 다른 대표적인 항산화 효소인 글루타티온 과산화 효소와 카탈라아제와의 비교 실험을 통해 퍼록시레독신-2가 동맥경화증 발병에 더욱 심각한 영향을 주는 것을 증명하였다. 마지막으로 항산화제인 ebselen을 투여하였을 때 퍼록시레독신-2 결핍에 의해 촉진되는 동맥경화증이 다시 억제되는 것을 확인함으로서 퍼록시레독신-2 결핍에 의한 동맥경화증 발병 촉진은 활성산소의 증가가 원인임을 확인할 수 있었다. 결론적으로, 퍼록시레독신-2는 혈관 및 면역세포에서 동맥경화증을 억제시키는 항산화 효소로서 퍼록시레독신-2의 활성 촉진과 같은 작용은 항염증 및 항동맥경화 치료제로서 유용한 방법이 될 수 있을 것으로 제안한다.