Role of macrophage enolase-1 in atherosclerosis progression

보호앙응우옛단 순천향대학교 일반대학원 2022 국내석사

Atherosclerosis, a dominant and growing cause of death and disability worldwide, is a chronic inflammatory disease of blood vessel walls. Macrophages are one of the critical factors in atherosclerosis progression and multiple approaches targeting inflammatory and metabolic pathways involving macrophages have sought to identify novel interventions to prevent and treat atherosclerosis. Recently, the glycolytic enzyme enolase-1 (ENO-1) has emerged as a novel regulator of cellular and inflammatory metabolism in certain pathologies. However, the role of macrophage ENO-1 in atherosclerosis has not yet been elucidated. In this study, we explored the role of ENO-1 in macrophage function and the impacts on atherosclerosis using two different macrophage cell lines and an atherogenic Ldlr-/- animal model. In both RAW264.7 macrophages and bone-marrow-derived macrophages (BMDMs), ENO-1 knockdown significantly suppressed the LPS-induced inflammatory response including production of the pro-inflammatory cytokines TNF-α and IL-6, NF-κB activation, and glycolysis. Interestingly, pro-inflammatory macrophage activation induced the enhancement of ENO-1 in the plasma membrane which is independent of the total ENO-1 protein level. Furthermore, treatment of the antagonist targeting the membrane-bound ENO-1 significantly downregulated Tnf, Il6, and Il1b mRNA in BMDMs. Concomitantly, antagonist-treated macrophages displayed the interference to the NF-κB signaling pathway but did not affect glycolytic activity. In vivo experiments with the atherogenic Ldlr-/- mouse model showed that ENO-1 antagonist-treated mice had smaller atherosclerotic lesion areas of the aorta than the control group. Furthermore, antagonist administration markedly reduced the circulating TNF-α, IL-6, and IL-1β levels. Nevertheless, the blood lipid profile remained uninterrupted. Moreover, the aortic macrophage population markedly decreased while neither the other immune cells including dendritic cells and T cells in the aorta nor the blood leukocytes population such as neutrophils, B cells, T cells, and monocytes did. Collectively, these results indicate that ENO-1 would be a critical factor in the pro-inflammatory macrophage activation and the development of atherosclerosis. More importantly, a unique anti-inflammatory antagonist can alleviate the production of pro-inflammatory cytokines when interacting with membrane ENO-1 and mitigate atherosclerosis progression in Ldlr-/- mice. Thus, membrane-bound ENO-1 may be a potential therapeutic target to prevent the progression of atherosclerosis.

TLR2 accelerates atherosclerosis through KLF4 in vascular smooth muscle cells

임주연 Graduate School, Yonsei University 2023 국내박사

Atherosclerosis is a chronic inflammatory disease that is caused by lipid accumulation in blood vessel walls, namely in macrophage foam cells and vascular smooth muscle cells. Accumulation of lipids induces an inflammatory response and accelerates vascular disease. Among the toll-like receptors (TLRs), TLR2 is an initiator and early inflammatory factor in atherosclerosis. Early atherosclerosis progresses due to lipid accumulation, leukocyte accumulation, and foam cell formation. Atherosclerosis is a vascular disease; however, studies on the function of TLR2 in vascular smooth muscle cells are insufficient. The activation of TLR2 signaling has an important role in promoting accumulation of lipids and phenotypic switching in vascular smooth muscle cells (VSMCs). Therefore, this study aimed to elucidate the effect of Krupple-like factor 4 (KLF4) regulation through TLR2 signaling, on the phenotype switching and acceleration of atherosclerosis in a cholesterol- treated vascular smooth muscle. Primary cells isolated from the whole aorta vascular smooth muscle cells of rats were treated with TLR2 siRNA and cholesterol (20 µg/ml) for 48 h. The expression of RNA and proteins associated with VSMC phenotype switching and signaling was analyzed using RT-PCR and western blotting. Caffeic acid phenylethyl ester (CAPE), an inhibitor of nuclear factor-kappa B (NF-kB) blocked the inflammation and phenotypic switching associated with KLF4 promoter binding. In this study, to investigate the acceleration of atherosclerosis and the underlying molecular mechanism of TLR2 action, a cholesterol-induced atherosclerosis mouse model was developed with overexpression of TLR2 specifically in the VSMCs. The mice were administered tamoxifen via intraperitoneal injection once every 24 hours for a total of 5 consecutive days and were fed a high-cholesterol diet to induce atherosclerosis over 8 weeks. The mouse model with the TLR2Tg SM22αCre+ ApoE-/- genotype showed lower expression of contractile markers (smooth muscle protein 22α, α-smooth muscle actin) and a higher expression of macrophage markers (CD68, Mac2) and KLF4, when compared to hat in SM22αCre+ ApoE-/- mice. In this study, to identify the acceleration of atherosclerosis, the molecular mechanism underlying TLR2 action was elucidated using an atherosclerosis disease model, in which the overexpression of TLR2 was induced specifically in vascular smooth muscle cells, and cholesterol was used for induction. Acceleration of TLR2 by cholesterol confirmed that KLF4 regulation through NF-kB activity affects VSMC phenotypic switching and the promotion of atherosclerosis. 죽상 동맥경화증은 만성 염증성 질환으로 혈관벽에 있는 플락이 혈관벽과 혈관 내피세포 및 혈관 평활근 세포내 지질 축적과 같은 합병증을 초래한다. 이러한 혈관의 지질 축적은 염증 반응을 유도하여 혈관 질환을 가속화시킨다. 이러한 TLRs의 발현 중 TLR2는 죽상 동맥경화증의 개시제 및 초기 염증 인자 이며, 지질 축적 및 백혈구 축적과 거품 세포 형성으로 인해 초기 죽상경화증 이 진행된다. 이러한 동맥경화증은 혈관 질환이지만 혈관 내피세포에서 활발 하게 연구가 진행되어 왔으며, 혈관 평활근 세포는 혈관벽의 구조적인 역할로 만 알려져 있다. 최근에는 죽상 동맥경화증에서의 혈관 평활근 세포에 대한 연구가 차지하는 비중이 높아지고 있는 추세이다. 하지만 죽상동맥경화증에서 의 혈관 평활근 세포에서 TLR2의 기능에 대한 연구는 부족한 실정이다. TLR2 신호전달의 활성화는 지질 축적 및 혈관 평활근 세포 표현형 전환을 촉진하는 데 중요한 역할을 한다. Rat 대동맥의 전체 혈관 평활근 세포에서 분리한 primary cell에서 TLR2 siRNA와 콜레스테롤을 48시간동안 처리하였다. 표현형 전환 및 신호 메커니즘과 관련된 RNA 및 단백질 발현을 RT-PCR 및 웨스턴 블롯으로 분석하였다. 또한 VSMC 표현형 전환의 중요 인자로 알려진 KLF4의 활성 및 발현을 증가시키는 NF-kB의 인산화를 확인하였다. NF-kB의 억제제인 카페산 페닐에틸 에스테르 (CAPE)는 염증 및 KLF4 및 표현형 전환을 억제하 였다. 본 연구에서는 죽상 동맥경화증의 촉진을 조사하기 위해 콜레스테롤 유도에 의한 TLR2의 분자생물학적 기전을 VSMC에서 특이적으로 TLR2 과 발현 이 유도되는 마우스 죽상동맥경화증 질환 모델을 사용하여 콜레스테롤에 의해 TLR2 과발현으로 인해 동맥경화 촉진이 가속화 됨을 확인하였다.총 5일동안 24시간 마다 복강내 주사를 통해 타목시펜을 투여하고 8주동안 죽상동맥경화증을 유발하기 위해 고콜레스테롤 식이를 공급하였다. 또한, 생체 내에서 TLR2Tg SM22αCre+ ApoE-/- 유전자형으로 생산된 마우스 모델에서 SM22αCre+ ApoE-/- 마우스와 비교했을 때, 수축성 마커 SM22α, αSM-actin의 낮은 단백질 발현과 대식세포 마커의 CD68, Mac2 및 KLF4 높은 단백질 발현 을 보였습니다. 또한, 콜레스테롤에 의해 증가된 TLR2가 NF-kB 활성을 통해 전사 인자인 KLF4의 발현을 증가시켜 혈관 평활근 세포 전환을 유도하였다.본 연구는 콜레스테롤 처리된 혈관 평활근 세포에서 콜레스테롤에 의해 TLR2 신호 가속화는 NF-kB 인산화를 통해 KLF4 조절이 VSMC 표현형 전환 및 죽상동맥경화증의 촉진에 미치는 영향을 조사하였다.

Lipoprotein-Extracellular Vesicle Complexes : Their Role in Atherosclerosis Progression

Jeong, Jea Sic 부산대학교 대학원 2025 국내박사

Atherosclerosis is a chronic inflammatory disease marked by lipid accumulation within arterial walls, leading to cardiovascular complications. In recent years, extracellular vesicles (EVs) have been recognized for their critical role in cellular communication and disease pathogenesis. Here, we characterize a novel subclass of EVs, termed ‘Lipo-EVs,’ enriched with lipoproteins and derived from liver cells under pathological conditions that resemble non-alcoholic fatty liver disease (NAFLD) and hepatitis. Using HepG2 liver cell models, we isolated Lipo-EVs and confirmed their structural integrity, characterized by a phospholipid bilayer and enriched lipoproteins (Apo A, Apo B, Apo E). Our findings suggest that these Lipo-EVs play a significant role in cholesterol transport, inflammation, and endothelial barrier disruption, which are central to atherogenesis. Through in vitro studies on HepG2 cells, HUVECs, and RAW264.7 macrophages, we found that Lipo-EVs bypass the regulatory LDL receptor (LDLR) pathway and promote continuous cholesterol uptake, contributing to lipid accumulation in liver cells. Unlike LDL or oxidized LDL (oxLDL), Lipo-EVs were associated with persistent cholesterol uptake, evident from unaltered LDLR expression and sustained lipid storage marker levels, which may accelerate fatty liver disease progression. Furthermore, Lipo-EVs impaired endothelial integrity by reducing the expression of tight junction proteins (Zonula Occludens-1 (ZO-1), Occludin), promoting increased permeability across the endothelial barrier. This permeability increase suggests enhanced cholesterol translocation to the subendothelial space, amplifying atherosclerotic plaque formation potential. In macrophages, Lipo-EVs promoted foam cell formation more efficiently than LDL or oxLDL at comparable cholesterol concentrations. Lipo-EVs exposure led to elevated ACAT1 expression, indicating increased cholesterol storage, while enhanced scavenger receptor expression (e.g., LOX1) allowed unregulated cholesterol uptake. Additionally, Lipo-EVs induced significant upregulation of pro-inflammatory markers (e.g., TNFα, IL-6), highlighting their role in inflammation. The elevated levels of reactive oxygen species (ROS) observed in macrophages exposed to Lipo-EVs suggest enhanced oxidative stress, further contributing to plaque instability and atherogenic progression. In vivo studies using a high-fat diet (HFD)-induced atherosclerosis model in mice revealed elevated Lipo-EV levels in blood, and an increase in plaque formation compared to LDL-treated controls. The Lipo-EV group showed intensified lipid deposition and reduced arterial elasticity, underscoring their impact on plaque instability and adverse cardiovascular outcomes. These findings indicate that Lipo-EVs exert unique effects on lipid metabolism, inflammation, and oxidative stress within cells central to atherosclerosis. Our results support the hypothesis that Lipo-EVs act as alternative cholesterol carriers independent of LDLR and contribute to atherosclerosis by altering cellular signaling, promoting lipid deposition, and enhancing inflammation. Targeting Lipo-EV production or inhibiting their uptake represents a potential therapeutic strategy for mitigating lipid-driven inflammation and plaque formation. Furthermore, Lipo-EVs may serve as biomarkers for assessing atherosclerotic burden, given their presence in circulation correlates with disease progression. As traditional cholesterol measures may not capture the lipid content within Lipo-EVs, incorporating these vesicles into clinical assessments could improve diagnostic accuracy and advance treatment strategies. Notably, these findings challenge the conventional view of HDL as inherently protective. When HDL is complexed within Lipo-EVs, it may contribute to cholesterol deposition and exacerbate atherosclerosis, suggesting that HDL’s cardioprotective role may vary depending on its association with specific vesicle structures. Further research into Lipo-EV-mediated pathways could reveal novel intervention points, aiding in the development of targeted therapies for atherosclerosis.

(The) roles of peroxiredoxin-2 in mouse atherosclerosis model

박종길 서울대학교 대학원 2012 국내박사

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. 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.

(The) roles of CD137 in mouse atherosclerosis

정인혁 서울대학교 대학원 2014 국내박사

The tumor necrosis factor receptor superfamily (TNFRSF), which includes CD40, LIGHT, and OX40, plays important roles in the development of atherosclerosis. CD137 (4-1BB), a member of the TNFRSF, is an activation-induced T cell co-stimulatory molecule. Signaling via CD137 up-regulates survival genes, enhances cell division, induces cytokine production, and prevents activation-induced cell death in T cells. CD137 has been reported to be expressed in human atherosclerotic plaque lesions. However, limited information is available on the precise role of CD137 in the development of atherosclerosis using mice models. To study the roles of CD137 in atherogenesis, we generated CD137 deficient apolipoprotein E knockout (ApoE-/-CD137-/-) mice and CD137 deficient low density lipoprotein receptor knockout (Ldlr-/-CD137-/-) mice. We found that CD137 is a significant atherosclerosis promoting factor mainly expressed on T cells infiltrated into atherosclerotic plaque lesions. Moreover, our finding showed that deficiency of CD137 strongly attenuates formation of atherosclerotic plaque lesion in ApoE-/- and Ldlr-/- mice, which implicates CD137 as a potential therapeutic target for atherosclerosis disease control. Atherosclerotic plaques contain blood-borne inflammatory and immune cells such as macrophages and T cells, as well as vascular cells. Formation of atherosclerotic plaque lesions are characterized by inflammation, lipid accumulation, cell death, and fibrosis. Adaptive immunity, in particular T cells, is highly involved in atherosclerosis. When the naive T cell encounters antigen-presenting cell such as macrophages and dendritic cells that presents an antigenic peptide including oxidized low density lipoprotein (oxLDL), T cells undergo activation and become effector T cells secreting Th1 cytokine such as interferon gamma (IFN-γ), which promotes the development of atherosclerosis. Enhanced T cell activation by co-stimulatory receptors significantly increases inflammatory cytokine release by monocyte/macrophages, further exacerbating inflammation and promoting atherosclerosis. Here, we studied functional mechanisms of CD137 in atherosclerosis. We found that CD137 functionally induces activation of T cell by generation of IFN-γ. In turn, IFN-γ induces monocytes/macrophages and endothelial cells to augment pro-inflammatory cytokine production through a positive feedback mechanism, thereby facilitating formation of atherosclerotic plaque. Over time, mature plaques advance to a vulnerable plaque, being more prone to rupture, causing subsequent atherothrombotic vascular disease such as myocardial infarction. Vulnerable plaques generally have a large necrotic core, and thin fibrous caps, attributable to the death of macrophages and vascular smooth muscle cells (VSMCs). Vulnerable plaques also contain elevated levels of proteases, including matrix metalloproteinases (MMPs), which might degrade the extracellular matrix and weaken fibrous caps. Our previous results suggested that CD137/CD137L signaling facilitates atherosclerosis. However, there is no report whether CD137 signaling affects plaque stability of advanced atheroma or not. To answer this question, we tried to elucidate whether CD137 can induce advanced plaque phenotype in atherosclerotic model mice. To solve the mechanism of CD137 in plaque stability, we focused on T cells, macrophages, and VSMCs, which are major cells involved in plaque stability. We found CD137 expression in these cells in atherosclerotic plaque lesion. Next, we investigated the functional mechanisms and signaling pathways of CD137 on these cells. Finally, we tried to confirm whether the in vivo activation of CD137 signaling using agonistic anti-CD137 mAb can exacerbate plaque stability in plaque of high fat diet fed Ldlr-/- mice. These observations could support the role of CD137 in stability of advanced atherosclerotic plaque. In summary, we show that 1) CD137 is a significant atherosclerosis-promoting factor mainly expressed on T cells infiltrated into atherosclerotic plaque lesions. Deficiency of CD137 reduces atherosclerosis in both normal chow diet fed ApoE-/- and high fat diet fed Ldlr-/- mice. 2) CD137 signaling induces activation of T cell by generation of IFN-γ, which leads to another phase of recruitment and activation of macrophages. The activated macrophages produce TNF-α and MCP-1, which cause endothelial CD137 expression. The endothelial CD137 signaling induces the production of MCP-1 and cell adhesion molecules, leading to enhancement of leukocyte recruitment to the atherosclerotic plaque. Therefore, CD137 signaling facilitates atherosclerosis through a positive feedback mechanism to T cells, macrophages, and endothelial cells. 3) In advanced atheroma, we found that CD137 induces advanced atherosclerotic plaque phenotype exhibiting enhanced plaque necrosis, decreased collagen and VSMCs content, and enhanced macrophage infiltration. CD137 facilitates T cell activation leading to the production of INF-γ, and increases macrophage derived MMP-9 through phosphorylation of p38 MAPK and ERK1/2 pathways. Moreover, activation of CD137 signaling inhibited Bcl-2, and then by up-regulated cleaved caspase-3, leads to apoptosis of VSMCs.

Newer biomarker-based prediction of atherosclerosis risk

이혁희 Graduate School, Yonsei University 2024 국내박사

서론: 안지오포이에틴-1은 혈관내피세포의 Tie-2 수용체와 결합하여 혈관의 안정성과 항상성을 증대하며, 안지오포이에틴-2는 위 작용을 억제한다. 이러한 분자생물학적 기전 상 안지오포이에틴-2, 안지오포이에틴-1 및 수용성 Tie-2는 죽상경화증 예측 바이오마커로 활용될 잠재력이 있다. 우리는 본 연구에서 (1) 안지오포이에틴 연관 바이오마커(안지오포이에틴-2, 안지오포이에틴-1 및 수용성 Tie-2) 연구를 위한 코호트를 구축하고, (2) 해당 바이오마커들의 기본 특성을 파악하며, (3) 해당 바이오마커들이 죽상경화증 위험도 예측에 도움이 될 수 있는지 확인하고, (4) 해당 바이오마커들을 활용한 죽상경화증 위험도 예측모형을 개발하고자 하였다. 연구방법: 2021년부터 2023년 사이 CMERC 및 CMERC-HI 코호트로부터 총 924명의 대상자가 모집되었으며, 검진을 통해 대상자들의 전통적 심혈관 위험인자(나이, 성별, 현재흡연, 수축기혈압, 혈압강하제 복용, 당뇨병, 총콜레스테롤, 고밀도지단백콜레스테롤 및 지질강하제 복용), 혈중 안지오포이에틴 연관 바이오마커 농도 및 관상동맥 석회화지수 등의 정보를 수집하였다. 죽상경화증은 중증 관상동맥 죽상경화증(관상동맥 석회화지수 >400)과 관상동맥 죽상경화증(관상동맥 석회화지수 >0)의 두 가지 지표로 정의되었다. 죽상경화증 예측 로지스틱 회귀모형의 성능은 C-지수, 호스머-레메쇼 카이제곱 통계치 및 일치도 그림 등의 방법으로 평가하였으며, 안지오포이에틴 연관 바이오마커에 의한 예측능 향상은 ΔC-지수, 연속형 순재분류향상 및 통합식별향상 등으로 평가하였다. 최종 개발된 죽상경화증 위험도 예측모형에 대해서는 내적 검증 및 홀드아웃 검증을 시행하였다. 연구결과: 총 924명의 대상자(나이 중위수, 62세; 60.5% 여성) 중 중증 관상동맥 죽상경화증은 60명(6.5%), 관상동맥 죽상경화증은 390명(42.2%) 에서 존재하였다. 안지오포이에틴 연관 바이오마커들은 서로 미약한 상관관계를 보였으며, 전통적 심혈관 위험인자와는 유의한 연관성을 보이지 않았다. 해당 바이오마커들은 전통적 심혈관 위험인자 기반 회귀모형의 중증 관상동맥 죽상경화증 위험도 예측력(ΔC-지수, 0.019-0.030; 연속형 순재분류향상, 0.46-0.53; 통합식별향상, 0.037-0.061)과 관상동맥 죽상경화증 위험도 예측력(ΔC-지수, 0.007-0.014; 연속형 순재분류향상, 0.18-0.20; 통합식별향상, 0.011-0.023)을 모두 향상시켰다. 최종 개발된 안지오포이에틴 기반 죽상경화증 위험도 예측모형은 내적 검증에서 우수한 식별력을 보였으며(C-지수 0.793-0.849), 홀드아웃 검증에서도 준수한 식별력을 보였다(C-지수 0.678-0.791). 일치도 그림 상 모형의 예측 위험도는 실제 위험도와 전반적으로 잘 일치하였다. 간편한 임상 적용을 위해 추가로 개발한 죽상경화증 위험도 점수 역시 좋은 식별력을 보였다(C-지수 0.790-0.837). 결론: 안지오포이에틴 연관 바이오마커들은 전통적 심혈관 위험인자 기반 회귀모형의 죽상경화증 위험도 예측력을 유의하게 향상시켰으며, 최종 개발된 안지오포이에틴 기반 죽상경화증 위험도 예측모형은 검증 결과 우수한 성능을 보였다. 본 연구에서 개발한 예측모형은 심혈관질환 일차예방을 위한 고위험 인구 선별 및 관상동맥 석회화지수 0인 대상자의 위험도 감시에 이용될 수 있을 것으로 기대된다. 안지오포이에틴 연관 바이오마커들이 임상 심혈관질환 사건 예측에도 활용될 수 있을지에 대해서는 추가적인 연구가 필요하다. Background: Angiopoietin-1 and its receptor, tunica interna endothelial cell kinase 2 (Tie-2), maintain endothelial integrity and homeostasis, while Angiopoietin-2 counteracts these effects. Given their biological actions, Angiopoietin-2, Angiopoietin-1, and soluble Tie-2 may have the potential to serve as predictive biomarkers for subclinical atherosclerosis. In this prospective ancillary study of the Cardiovascular and Metabolic Diseases Etiology Research Center (CMERC) and CMERC High-Risk (CMERC-HI) cohorts, we aimed to (1) construct a biomarker subcohort for the exploration of Angiopoietin-related biomarkers (i.e., Angiopoietin-2, Angiopoietin-1, and soluble Tie-2); (2) delineate the baseline characteristics of these biomarkers; (3) examine whether these biomarkers improve risk prediction for subclinical atherosclerosis beyond traditional risk factors; and (4) develop and validate subclinical atherosclerosis risk prediction models using these biomarkers. Methods: We enrolled a total of 924 participants from the CMERC and CMERC-HI cohorts between 2021 and 2023. Data on traditional risk factors, including age, sex, current smoking, systolic blood pressure, blood pressure-lowering drug use, diabetes, total and high-density lipoprotein cholesterol levels, and lipid-lowering drug use, were collected at baseline. Serum levels of Angiopoietin-related biomarkers were measured using enzyme-linked immunosorbent assay from the blood samples collected at baseline. The coronary artery calcium (CAC) was measured by cardiac computed tomography; the CAC score was calculated using the Agatston method. The primary measures of subclinical atherosclerosis were severe coronary atherosclerosis (defined as a CAC score >400) and coronary atherosclerosis (defined as a CAC score >0). The predictive performance of the models was evaluated using the Harrell’s C-index, Hosmer-Lemeshow chi-square statistic, and calibration plot. The additive utility of Angiopoietin-related biomarkers beyond traditional risk factors was assessed using the ΔC-index, continuous net reclassification improvement (cNRI), and integrated discrimination improvement (IDI). We developed subclinical atherosclerosis risk prediction models incorporating Angiopoietin-related biomarkers (Angiopoietin-based prediction models) and performed internal and holdout validation. Results: Of the 924 participants (median age, 62 years; 60.5% female), severe coronary atherosclerosis (CAC score >400) and coronary atherosclerosis (CAC score >0) were present in 60 (6.5%) and 390 (42.2%) participants, respectively. Angiopoietin-related biomarkers showed a poor correlation with each other and weak or no association with traditional risk factors. Angiopoietin-related biomarkers significantly improved risk discrimination and stratification for both severe coronary atherosclerosis (ΔC-index, 0.019-0.030; cNRI, 0.46-0.53; IDI, 0.037-0.061) and coronary atherosclerosis (ΔC-index, 0.007-0.014; cNRI, 0.18-0.20; IDI, 0.011-0.023) when added to traditional risk factor-based models. The Angiopoietin-based prediction models showed good discrimination in internal validation (optimism-corrected C-index, 0.793-0.849) and decent discrimination in holdout validation (C-index, 0.678-0.791). The predicted probabilities were in good agreement with the observed probabilities. Integer risk scores for subclinical atherosclerosis were also developed and showed satisfactory performance (C-index, 0.790-0.837). Conclusion: Angiopoietin-related biomarkers significantly improved risk prediction for subclinical atherosclerosis when added to traditional risk factor-based models. Angiopoietin-based prediction models, as well as integer risk scores, showed good predictive performance. The newly developed prediction models are expected to be utilized for identifying high-risk populations for the primary prevention of cardiovascular disease (CVD) and for surveilling individuals with a CAC score of 0. Whether Angiopoietin-related biomarkers improve risk prediction for clinical CVD events needs to be determined in future studies.

A Study on Atherosclerosis of Common Carotid Artery in Korean Men

TRAN THI THANH LOAN 성균관대학교 일반대학원 2012 국내박사

Since the mid-1990s carotid intima-media thickness (IMT) by B-mode ultrasound has increasingly been used for cardiovascular disease. It has also been used as a surrogate endpoint to monitor the efficacy of therapy against atherosclerosis in clinical trials.However, regression or slowed progression of carotid IMT induced by cardiovascular drug therapies, did not reflect reduction in cardiovascular events and current consensus guidelines are not available on which site and how carotid IMT sampling should be made. Therefore, it is still under debate that carotid IMT can be used as an independent risk factor, especially in younger subjects with intermediate risk. On the other hand, it has been well established that collagen and elastin are the most abundant extracellular matrix proteins of the arterial wall. They are responsible for the characteristic mechanical properties?tensile strength and elasticity. In this regard, both collagen and elastin may have considerable values in diagnostic information to understand the association of the major ECM proteins with atherosclerosis progression. Many previous studies focused on relationship between IMT and cardiovascular events, but histological analyses are few. Accordingly, I performed this study to assess that the IMT is really a good surrogate of atherosclerosis and a predictor of cardiovascular events by measuring the exact intima thickness and media thickness from postmortem tissue sections of the common carotid artery. To evaluate this, postmortem materials of the whole of both the left and the right common carotid artery from 133 Korean men aged from 20 to 78 years were enrolled for histopathology and computer-assisted morphometry. Atherosclerosis-associated collagen and elastin were quantitatively analyzed. Correlation coefficients of IMT were smaller than those of intima thickness but IMT was well associated with age, atherosclerosis score, plaque area, total cholesterol, low-density lipoprotein cholesterol and triglyceride. Moreover, when IMT was thicker than 1 mm, possibility of carotid atherosclerosis accompanied with coronary artery stenosis and coronary calcification was much higher. In addition, serum lipids were assayed to assess their relationship with IMT. Quantitative analyses of collagen and elastin were also made to assess relationship between progression of atherosclerosis and plaque area, i.e., nodular accumulation of soft, flaky lipid deposit with smooth muscle cells, macrophages, leukocytes, and extracellular matrix components, with or without fibrous cap. And collagen tended to increase while elastin tended to decrease as atherosclerosis score increased; collagen increased and elastin decreased when compared plaque to plaque-free area in the same segment, Taken together, these findings support that the carotid IMT can be used as a good surrogate marker of atherosclerosis and predictor of coronary heart disease. And plaque formation may influence significant quantitative changes in collagen and elastin.

The Role of Dietary Fats in Driving Atherosclerosis

Wang, Zenan Yale University ProQuest Dissertations & Theses 2023 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Cardiovascular diseases (CVDs) are the number one killer of people worldwide, of which coronary heart disease, which is mainly due to atherosclerosis, is the main contributor to death. Nonetheless, the etiology of atherosclerosis remains poorly understood. It is universally accepted that dietary fat influences atherosclerosis, however, the role of specific fats is not well-understood. Despite the widely-held perception that saturated fats drive atherosclerotic CVDs while monounsaturated fats are protective, there have been numerous studies from human and mice that do not fit with this prevailing narrative. Thus, despite the importance of dietary fats in CVDs, the exact effects remain controversial and there is a substantial gap in knowledge about how specific dietary fats drive atherosclerotic CVDs.In my thesis work, I systematically dissect the role of dietary fats in atherosclerosis using an unbiased diet screen. Mice were fed a series of custom isocaloric high-fat diets (HFDs), which differed only in the fat source, for 12 weeks and atherosclerosis progression was assessed. I found that dietary fat composition significantly influences the extent of atherosclerosis. Lipidomic analysis of the diets revealed that atherosclerosis correlates with only two dietary fatty acids: C16:0, palmitic acid (PA) and C18:1 n-9, oleic acid (OA). This unbiased approach, for the first time, elucidates precisely that it is not fatty acids per se, but dietary PA and OA that play critical roles in atherosclerosis.Leveraging the correlative relationship, I characterized the atherosclerotic plaque morphology developed in mice fed the high-OA and high-PA diets, which indicates differential regulation of lesional macrophage apoptosis based on dietary fat composition despite comparable plaque size. This suggests that dietary PA and OA drive atherosclerosis by different mechanisms. In vitro transcriptome analysis of fatty acid-treated macrophages revealed that PA and OA induce two separate pathways that are known to be involved in human atherosclerosis, with PA inducing macrophage colony stimulating factor (M-CSF, or CSF1) and OA downregulating liver X receptor (LXR).While both CSF1 and LXR pathways are known to affect atherosclerosis, their roles in response to dietary PA and OA are unknown. Therefore, I determine the role of PA and OA-induced pathways in atherosclerosis by targeting these pathways in vivo both individually and in combination in the context of high-PA and high-OA diets. I found that the efficacy of targeting each pathway individually is diet-dependent, supporting the central premise that there are two independent diet-induced atherosclerotic pathways. Incredibly, given that PA and OA are always present together in the diet and in vivo, targeting both OA and PA-induced pathways simultaneously is found to have a profound, synergistic effect on ameliorating atherosclerosis.Together, these data indicate that there are two separate dietary fatty acid-induced pathways that drive atherosclerosis.

Association of visceral fat thickness with carotid atherosclerosis and inflammation in peritoneal dialysis patients

이미정 Graduate School, Yonsei University 2011 국내석사

Background: Patients with advanced chronic kidney disease (CKD) requiring maintenance dialysis have a significantly higher risk of cardiovascular mortality. In general population, anthropometric measurements of central obesity such as high body mass index (BMI), waist circumference (WC), and waist-to-hip ratio (WHR) are associated with increased cardiovascular mortality. However, the association between obesity and clinical outcomes in patients with peritoneal dialysis (PD) is conflicting. The aim of this study was to investigate whether visceral fat thickness had predictive role in carotid atherosclerosis determined by carotid intima-media thickness (cIMT) in PD patients. Methods: A cross-sectional study was undertaken in 88 prevalent PD patients between February 2010 and July 2010. BMI, WC, and WHR were measured as anthropometric indices of obesity. Visceral fat thickness (VFT) and subcutaneous fat thickness (SFT) were determined by sonographic measurement of abdominal fat. cIMT was measured as a surrogate marker of atherosclerosis. Carotid atherosclerosis was defined as cIMT ≥1 mm. Results: The mean age was 53.3 ± 11.8 years and the mean duration of PD was 63.6 months. Fifty-four patients (61.4%) were male and 22 patients (25%) were diabetes. The mean cIMT was 0.76 ± 0.26 mm, and the prevalence of carotid atherosclerosis was 20.4% (18/88). Patients with carotid atherosclerosis had significantly higher WC (89.4 ± 9.9 vs. 83.7 ± 7.8 cm, p = 0.033), higher WHR (0.94 ± 0.05 vs. 0.91 ± 0.05, p = 0.038), and higher VFT (42.5 ± 15.1 vs. 32.2 ± 8.9 mm, p <0.001) compared to patients without carotid atherosclerosis. In univariate analysis, VFT [Odds ratio (OR) = 1.085, 95% Confidence interval (CI): 1.032-1.14, p = 0.001], WHR (OR = 1.105, 95% CI: 1.033-1.218, p = 0.043), BMI (OR = 1.24, 95% CI: 1.029-1.494, p = 0.024), and WC (OR = 1.085, 95% CI: 1.016-1.158, p = 0.014) were significant risk factors of carotid atherosclerosis. However, multivariate logistic regression analysis revealed VFT was a single independent factor associated with carotid atherosclerosis after adjustment of demographic and biochemical parameters (OR = 1.089, 95% CI: 1.021-1.161, p = 0.009). Moreover, VFT remained as an independent risk factor of carotid atherosclerosis in multivariate logistic regression analysis for comparison of relative independent association of obesity indices with carotid atherosclerosis (OR = 1.095, 95% CI: 1.004-1.194, p = 0.04). The respective areas under the receiver operating characteristic curve (AUC) of VFT was higher than the AUCs of all other parameters (VFT; AUC 0.705, p=0.007). When the patients were divided into three groups according to VFT, patients with high VFT tertile showed higher inflammatory markers such as high sensitivity C-reactive protein and fibrinogen, and higher insulin resistance compared to other groups. Conclusion: The main finding of this study is VFT, not SFT, is more predictive for carotid atherosclerosis than all other obesity indices in PD patients. Although WC and WHR showed significant relationships with carotid atherosclerosis in univariate analysis, VFT measured by abdominal ultrasonography was an independent risk factor of carotid atherosclerosis after adjustment for confounding variables. Therefore, sonographic measurement of VFT could be useful to stratify the risk of cardiovascular outcomes in PD patients.

신부전 유도 아포지단백질 E결핍 마우스에서 라파마이신과 아토바스타틴의 병용 투여 후 동맥경화 및 지질대사 개선 효과

안상현 서울대학교 대학원 2023 국내박사

배경 및 목적: 만성신부전은 전신적인 pro-inflammatory 상태로 죽경화증의 발생이 증가하여 주요 심혈관계 질환이 증가한다. 라파마이신(rapamycin)은 죽경화증의 억제 작용을 하는 것으로 알려져 있으나 이상지혈증을 유발할 수 있다. 따라서 아토바스타틴(atorvastatin)과 같은 지질조절제(lipid-lowering agents)의 병용 투여를 권장한다. 그럼에도 불구하고 만성신부전에서 라파마이신과 아토바스타틴의 병용투여의 효과에 대한 연구는 없다. 본 연구에서는 신부전이 유도된 아포지단백질 E결핍(apolipoprotein E-deficient, ApoE-/-)에서 라파마이신과 아토바스타틴의 병용 투여가 죽경화증 발생과 혈중 지질대사에 미치는 영향을 알아보고자 한다. 방법: 마우스를 5개 군으로 무작위로 나눈 후 1개 군에서 헛수술(sham-operation)과 4개 군에서 5/6에 해당하는 신장절제를 시행하여 신부전을 유도했다(신부전군, 아토바스타틴군, 라파마이신군, 라파마이신+라파마이신군). 아토바스타틴(10 mg/kg)이나 라파마이신(0.5 mg/kg)을 해당 군에 10주 동안 매일 경구 위영양(oral gavage)했다. 죽경화증 병변의 확인을 위해 대동맥과 대동맥굴(aortic sinus)를 Oil Red O염색했다. 간에서 qRT-PCR과 Western blot을 이용하여 지질대사 유전자와 단백질의 발현을 확인했다. 대동맥과 비장에서 pro/anti-inflammatory cytokine의 유전자의 발현정도를 측정했다. 결과: 라파마이신군과 라파마이신+아토바스타틴군의 대동맥과 대동맥굴의 Oil Red O염색에서 신부전군과 비교하여 의미 있는 죽경화증 발생의 감소가 관찰되었다. 하지만 아토바스타틴군에서 죽경화증 발생 감소는 없었다. 라파마이신과 아토바스타틴의 병용 투여는 혈청의 지질 프로필을 향상하였고 간에서 콜레스테롤 수송(ABCG5), 담즘합성(CYP7A1) 그리고 지질대사(PPARγ, ApoA1)와 연관된 유전체의 발현이 증가했다. 신부전군은 비장에서 TNF-α, IL-6and IL-1β과 대동맥에서 TNF-α and IL-4의 atherosclerosis-promoting cytokines의 유전체의 발현이 증가했다. 라파마이신의 투여는 이들 유전자의 감소와 연관이 있었다. 아토바스타틴군과 라파마이신+아토바스타틴군은 비장에서 TNF-α와 IL-1β를 감소시켰다. 결론: 본 연구는 신부전 유도 ApoE-/-마우스에서 라파마이신은 inflammatory cytokine의 발현의 조절을 통해 의미 있게 죽경화증의 발생을 감소시켰고 아토바스타틴의 병용 투여는 추가적인 죽경화증의 발생을 억제하지는 않았으나 지질 대사를 향상시켰다. Background: Atherosclerosis arising from the pro-inflammatory conditions associated with chronic kidney disease (CKD) increases major cardiovascular morbidity and mortality. rapamycin is known to inhibit atherosclerosis under CKD and non-CKD conditions, but it can cause dyslipidemia; thus, the co-application of lipid-lowering agents is recommended. Atorvastatin has been widely used to reduce serum lipids levels, but its synergic effect with rapamycin in CKD remains unclear. Here, we analyzed the effect of their combined treatment on atherosclerosis stimulated by CKD in apolipoprotein E-deficient (ApoE-/-) mice. Methods: The mice were randomly assigned to five groups, including one group with normal renal function (sham-operated) and the others with surgically induced CKD (by 5/6 nephrectomy) (vehicle vs. atorvastatin vs. rapamycin vs. rapamycin + atorvastatin). atorvastatin (10 mg/kg) and rapamycin (0.5 mg/kg) were administered by daily oral gavage for 10 weeks. Aorta and aortic sinus were stained with Oil Red O to compare the size of atherosclerotic lesions. The expression levels of lipid metabolism-related genes and proteins in the livers was evaluated by qRT-PCR and Western blot. The expression level of pro/anti-inflammatory cytokines in aorta and spleen was measured and compared using qRT-PCR. Results: Oil Red O staining revealed that treatment with rapamycin and rapamycin +atorvastatin, but not atorvastatin alone, significantly decreased the atherosclerotic lesions in the aorta and aortic sinus, compared to those seen in the control (CKD) group. The co-administration of rapamycin and atorvastatin improved the serum lipid profile and enhanced the liver expression levels of genes involved in cholesterol transport (ABCG5), bile acid biosynthesis (CYP7A1), and lipid metabolism (PPARγ, ApoA1). The CKD group showed increased levels of various genes encoding atherosclerosis-promoting cytokines in the spleen (TNF-α, IL-6 and IL-1β) and aorta (TNF-α and IL-4), and these increases were attenuated by rapamycin treatment. atorvastatin and rapamycin + atorvastatin decreased the levels of TNF-α and IL-1β in the spleen, but not in the aorta. Conclusion: These results indicate that, in CKD-induced ApoE-/- mice, rapamycin significantly reduces the development of atherosclerosis by regulating the expression of inflammatory cytokines and the co-application of atorvastatin improves lipid metabolism.