PPARδ/IL-10 경로를 통한 c-Met 억제제의 항염증 효과가 동맥경화의 억제에 미치는 영향에 관한 연구

학위논문(박사) -- 서울대학교대학원 : 의과대학 의학과, 2023. 2. 이태승.Background: Atherosclerosis is a leading cause of cardiovascular disease and the adhesion of inflammatory cells to intimal endothelial cells is a well-known mechanism of atherosclerosis progression. Tyrosine kinases play an important role in inflammation and strategies to control tyrosine kinase activity have been widely used to modulate chronic inflammatory states. In this study we investigated the impact of a c-Met inhibitor capmatinib (CAP) on the suppression of atherosclerotic inflammatory response by investigating CAPs impact on 1) lipopolysaccharide (LPS)-mediated adhesion of human umbilical vein endothelial cells (HUVECs) and THP-1 monocytes in vitro, and 2) progression of atherosclerosis in ApoE knockout mice in vivo. Methods: HUVECs and THP-1 monocytes were treated with LPS and CAP. Protein expression levels related to endothelial cell adhesion and inflammation were determined using Western blotting. Target protein (PPARδ, IL-10) knockdown was conducted using small interfering (si) RNA transfection. Adhesion between HUVECs and THP-1 cells was assayed using green fluorescent dye. CAPs effect in vivo were tested on ApoE (-/-) mouse fed with western diet. CAP was fed orally for 5 weeks and compared with control, while PPARδsiRNA injection was performed every other day for 5 weeks in another group to verify the PPARδ-related mechanism of CAP. Results: Through in vitro studies, we found that CAP treatment suppressed cell adhesion between THP-1 monocytes and HUVECs and the expression of adhesive molecules, such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Moreover, phosphorylation of inflammatory markers, such as NFκB and IκB as well as TNFα and monocyte chemoattractant protein-1 (MCP-1), released from HUVECs and THP-1 monocytes, was decreased by CAP treatment. Treatment with CAP increased PPARδ and IL-10 expression, while siRNA-associated suppression of PPARδ and IL-10 attenuated the effects of CAP on cell adhesion between HUVECs and THP-1 cells and inflammatory responses. Furthermore, PPARδ siRNA suppressed CAP-mediated induction of IL-10 expression. In vivo, CAP abolished the increased expression of ICAM-1, E-selectin and TNFα in ApoE(-/-) atherosclerotic mice, and increased the expression of anti-inflammatory marker IL-10 as well as PPARδ. These effects were reversed in the PPARδ siRNA transfected mouse group. Conclusion: These findings imply that CAP improves inflamed endothelial-monocyte adhesion in vitro via a PPAR/IL-10-dependent pathway, and this pathway was verified in vivo in an atherosclerotic mouse model. The current study provides insights for a new therapeutic approach in treating atherosclerosis.배경: 동맥경화는 심혈관질환의 주요 원인으로 알려져 있으며, 염증세포의 혈관 내피세포 안착은 동맥경화의 진행에 있어 매우 중요한 기전이다. Tyrosine kinase는 염증 반응에 중요한 역할을 하는 것으로 알려져 있으며, 이에 tyrosine kinase을 조절하는 전략을 통해 다양한 만성 염증성 질환을 치료하고 있다. 본 연구에서는 c-Met inhibitor인 capmatinib (CAP)의 동맥경화성 염증반응 억제 효과를 확인하기 위하여 1) LPS에 의해 유발되는 인간 탯줄정맥 내피세포 (HUVEC)와 THP-1 단핵구의 부착에 대한 억제 효과를 확인하고, 2) 동맥경화 모델인 ApoE knockout mouse에서 동맥경화의 억제 효과를 확인하고자 한다. 방법: HUVEC과 THP-1 단핵구를 배양하여 LPS처리 후 CAP를 투여하였다. 이에 따른 단백질 구현은 Western blot을 통해 확인하였으며, 목표 단백질의 녹다운 (knockdown)을 위해 짧은 간섭 RNA (siRNA) 형질주입(transfection) 기법을 사용하였다. HUVEC과 THP-1 세포간 부착 여부는 초록 형광 염색을 이용하여 확인하였다. CAP의 효과를 in vivo에서 확인하기 위해 ApoE knockout mouse에 western diet을 5주간 복용시키고, 1개 군에서는 CAP를 경구로 복용시켰으며, 다른 1개 군에서는 CAP를 경구로 복용시킴과 동시에 PPARδsiRNA를 2일에 한번 꼬리정맥을 통해 주입하였다. 결과: In vitro에서는 CAP에 의해 HUVEC과 THP-1 세포의 부착이 억제되었으며, 이 과정에서 세포부착분자인 ICAM-1, VCAM-1 및 E-selectin의 감소가 확인되었다. 또한 CAP에 의해 NFκB, IκB, TNFα 및 MCP-1과 같은 염증 관련 인자들의 억제 효과를 HUVEC과 THP-1 세포에서 확인하였다. 또한 CAP에 의해 PPARα 및 IL-10 표현의 증가를 확인하였으며, si-RNA에 의해 PPARα 및 IL-10을 억제시킨 경우 CAP의 효과 (HUVEC 및 THP-1 세포 부착 및 염증 반응 억제)가 소실하였다. 아울러 CAP로 유도된 IL-10 표현이 PPARα siRNA에 의해 감소하는 현상을 확인하였다. In vivo에서는 ApoE (-/-) 동맥경화 쥐에서 증가되어 있던 ICAM-1, E-selectin 및 TNFα가 CAP 투여로 감소 효과를 확인하였으며, 반대로 항염증반응 표지자인 IL-10은 증가 소견을 보였다. 이런 효과는 PPARδ siRNA를 주입한 쥐에서는 소실되는 효과를 확인하였다. 결론: 위 결과에서 CAP는 염증으로 유발된 혈관내피세포와 단핵구의 부착을 억제시키는 효과가 있음을 확인하였으며, 이 과정이 PPARα/IL-10 경로가 관여함을 확인하였다. 본 연구를 통해 c-Met inhibitor의 동맥경화 억제 효과와 작용기전을 규명하였으며, 추후 동맥경화의 유효한 치료 전략으로 활용 가능할 것으로 기대된다.Chapter 1. Introduction 1 Chapter 2. Materials and Methods 4 Chapter 3. Results 9 Chapter 4. Discussion 13 Chapter 5. Conclusion 17 Bibliography 18 Figures 23 Abstract in Korean 40박

근육줄기세포의 생체 외 분화 유도 및 복부 대동맥류 형성 억제에 대한 연구

학위논문 (석사)-- 서울대학교 대학원 : 임상의과학과, 2013. 2. 이태승.Introduction: Abdominal aortic aneurysms (AAA) are a growing problem worldwide, yet there is no known medical therapy. The pathogenesis involves degradation of the elastic lamina by two combined mechanisms: increased degradation of elastin by matrix metalloproteinases (MMP) and decreased formation of elastin due to apoptosis of vascular smooth muscle cells (VSMC). In this study, we set out to examine the ability of muscle-derived stem cells (MDSC) to differentiate to VSMCs in vitro, and their potential role in the attenuation of AAA formation by inhibition of these pathogenetic mechanisms. Methods: Muscle-derived stem cells were isolated from murine skeletal muscles using a modified preplate technique. These cells were stimulated with PDGF-BB in vitro for differentiation to VSMC-like progenitor cells (VSMC-PC) and were subsequently implanted into elastase-induced AAAs in rats. After 6 weeks, the aortas were harvested and the formation of AAA was investigated. Immunohistochemical staining, RT-PCR, western blot and gel zymography for MMPs were performed and compared to a control group. Results: Isolated MDSCs showed characteristic expression of markers Sca-1 and CD34. When stimulated in vitro with PDGF-BB, the cells showed expression of α-SMA, a specific marker for smooth muscle cells. In vivo studies of elastase-perfused AAAs showed that the cell therapy group had decreased rate of aneurysm formation compared to control (83% vs. 50%), and MMP expressions at the genetic, protein and enzymatic levels were significantly decreased in the cell therapy group. Furthermore, direct implantation of VSMC-PCs in the intima of harvested aortas was visualized under immunofluorescent staining, suggesting that these cells were responsible for the inhibition of MMPs and consequent attenuation of AAA formation. Conclusions: These results show a promising role of stem cell therapy for the treatment of AAAs, and with further studies, may be able to reach clinical significance.Introduction 1 Materials and Methods 4 Results 11 Discussion 26 References 30 Abstract in Korean 36Maste