Therapeutic potential of endothelial colony‐forming cells in ischemic disease: Strategies to improve their regenerative efficacy

Cardiovascular disease (CVD) comprises a range of major clinical cardiac and circulatory diseases, which produce immense health and economic burdens worldwide. Currently, vascular regenerative surgery represents the most employed therapeutic option to treat ischemic disorders, even though not all the patients are amenable to surgical revascularization. Therefore, more efficient therapeutic approaches are urgently required to promote neovascularization. Therapeutic angiogenesis represents an emerging strategy that aims at reconstructing the damaged vascular network by stimulating local angiogenesis and/or promoting de novo blood vessel formation according to a process known as vasculogenesis. In turn, circulating endothelial colony‐forming cells (ECFCs) represent truly endothelial precursors, which display high clonogenic potential and have the documented ability to originate de novo blood vessels in vivo. Therefore, ECFCs are regarded as the most promising cellular candidate to promote therapeutic angiogenesis in patients suffering from CVD. The current briefly summarizes the available information about the origin and characterization of ECFCs and then widely illustrates the preclinical studies that assessed their regenerative efficacy in a variety of ischemic disorders, including acute myocardial infarction, peripheral artery disease, ischemic brain disease, and retinopathy. Then, we describe the most common pharmacological, genetic, and epigenetic strategies employed to enhance the vasoreparative potential of autologous ECFCs by manipulating crucial pro‐angiogenic signaling pathways, e.g., extracellular‐signal regulated kinase/Akt, phosphoinositide 3‐kinase, and Ca2+ signaling. We conclude by discussing the possibility of targeting circulating ECFCs to rescue their dysfunctional phenotype and promote neovascularization in the presence of CVD

혈장 유래 세포밖소포체 마이크로RNA 기반 소아 모야모야병의 진단 바이오마커 발굴

학위논문(박사) -- 서울대학교대학원 : 의과대학 의학과, 2022. 8. 김승기.Background. Moyamoya disease (MMD) is a chronic occlusive cerebrovascular disease known to be a major cause of stroke in children. Emerging evidence suggests that circulating extracellular vesicles (EVs) containing miRNAs in cerebrovascular disease plays a significant role in intercellular communication by delivering RNA cargo involved in biological processes. This study aimed to investigate the specific miRNAs loaded into MMD plasma-derived EVs, followed by identification of their roles and mechanisms. Methods. Plasma-derived EVs were isolated from normal control and MMD patients. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, ExoView and western blot. Profiling of miRNAs in EVs were determined using NanoString nCounter miRNAs analysis system and validated using ExoView and RT-qPCR. Endothelial colony forming cells (ECFCs) from MMD were isolated and the miRNA inhibitor was transfected to assess the cell viability, guanosine triphosphatase (GTPase) activity, and tubule formation. Results. There was no significant difference in size and distribution of EVs between normal and MMD EVs. However, the total number was higher, with CD81 and CD9 were lower and CD63 was higher in MMD EVs. miRNA profiling demonstrated that miR-512-3p were significantly upregulated in MMD EVs. Target prediction analysis of miR-512-3p showed that Rho guanine nucleotide exchange factor 3 (ARHGEF3) was downregulated by miR-512-3p in MMD ECFCs. Inhibition of miR-512-3p in ECFCs reduced miR-512-3p in EVs as well as ECFCs, and increased ARHGEF3 expression. The increase in ARHGEF3 through the inhibition of upregulated miR-512-3p in MMD EVs, which can restore dysfunction of tubule formation by activating GTPase. Conclusion. Our study implies that MMD EV miR-512-3p is correlated with the metabolism of ECFCs and may affect defective angiogenesis.배경: 모야모야병은 뇌혈관의 협착이 진행하는 질환으로 소아에서 뇌졸중의 주 원인으로 알려져 있다. 세포밖소포체에 탑재된 마이크로리보핵산이 세포간 전달에 있어 중요한 역할을 하고 질병의 발생과 진행에 영향을 미친다는 연구들이 보고되고 있다. 본 연구에서는 소아 모야모야병 환자의 혈장에서 유래된 세포밖소포체 탑재 마이크로리보핵산에 대해 분석하고, 이 질환의 병태생리에서 어떤 역할을 하는지 살펴보고자 한다. 방법: 소아 모야모야병 환자 및 젊은 건강한 성인을 대상으로 말초혈액을 채취하여 혈장을 분리하고, 혈장에서 세포밖소포체를 분리하였다. 정상군과 비교하여 환자군에서 차별 발현하는 세포밖소포체 내의 마이크로리보핵산을 확인하고, 환자 혈액에서 배양한 혈관내피전구세포의 메신저리보핵산 발현 결과와 매칭하여 해당 마이크로리보핵산이 표적하는 유전자를 확인하였다. 혈관내피전구세포에서 이 마이크로리보핵산의 억제제를 처리할 때 세포의 생존능, 표적 유전자의 발현 정도, 혈관형성능, GTPase 활성 정도의 변화를 확인하였다. 결과: 혈장 유래 세포밖소포체를 특성화분석 했을 때 정상군, 환자군 모두 세포밖소포체의 특성에 차이가 없음을 확인하였다. 마이크로핵산의 발현을 분석했을 때 정상군과 비교하여 환자군에서 miR-512-3p가 가장 높게 발현하였다. 이 마이크로핵산의 발현 량으로 수신자 조작 특성 곡선을 그리고 곡선 아래 면적을 분석했을 때 0.823로 모야모야병의 진단 바이오마커로서의 활용 가능성을 확인하였다. miR-512-3p의 표적 유전자 중 환자 혈관내피전구세포에서 가장 낮게 발현한 것은 ARHGEF3였다. 혈관내피전구세포에 miR-512-3p의 억제제를 처리하였을 때 세포의 생존능에는 영향을 주지 않았고, ARHGEF3의 발현이 증가했으며, 혈관 형성, GTPase 활성도 증가하였다. 결론: 소아 모야모야병 환자의 혈장 유래 세포밖소포체 탑재 miR-512-3p는 ARHGEF3를 억제하여 혈관내피전구세포의 혈관형성능에 영향을 준다.Abstract i Contents iii List of Figures and Tables iv List of Abbreviations v Introduction １ Materials and Methods ３ Results 11 Discussion 27 Conclusion 31 References 32 Abstract in Korean 35박