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New Therapeutic Approaches for Pulmonary Hypertension Using Human Umbilical Cord Blood-derived Mesenchymal stem cells and Modafinil

Title
New Therapeutic Approaches for Pulmonary Hypertension Using Human Umbilical Cord Blood-derived Mesenchymal stem cells and Modafinil
Authors
이혜련
Issue Date
2016
Department/Major
대학원 의과학과
Publisher
이화여자대학교 대학원
Degree
Doctor
Advisors
서석효홍영미
Abstract
BACKGROUND: Pulmonary arterial hypertension (PAH) is a refractory disease and causes right ventricular failure due to a progressive increase in pulmonary vascular resistance. In spite of current various therapeutic approaches, most treatment options generally have showed just minor improvements on the disease. Therefore, it is necessary to develop new therapies for PAH. The objective of this study were to investigate the effect of human umbilical cord blood derived-mesenchymal stem cells (hUCB-MSCs) and modafinil. ETHODS: This research was preceded in two parts. In the first part, the rats were grouped in the following manner: the C group (control), the M group (subcutaneous injection of monocrotaline (MCT) 60 mg/kg), the U group (hUCB-MSCs, 3 ×106 cells/rat transfusion). The U group received hUCB-MSCs transfusions through the external jugular vein one week after MCT injection. The rats were sacrificed at weeks 2, 3 and 4. Hemodynamics was measured by placing the catheter into the jugular vein in the anesthetized state. Pulmonary pathologies were assessed by measuring medial wall thickening pulmonary arterioles and the number of intra-acinar pulmonary arterioles. mRNA levels were evaluated by PCR. Protein expression levels were evaluated by western blot analysis or immunohistochemical staining. Cytokine assay and proliferation assay were also processed. In the second part, the rats were grouped as follows: the C group, the M group (subcutaneous injection of MCT 60 mg/kg), the MD group was subdivided into 3 subgroups (oral administration of modafinil, 4, 10, 50 mg/kg/day). Evaluation methods of hemodynamics, pulmonary pathologies, Protein expression levels and immuhohistochemical staining are same as described above. Pulmonary arterial smooth muscle cells (PASMCs) were enzymatically isolated from pulmonary arteries at 37 ℃. Intracellular cyclic adenosine monophosphate (cAMP) production was measured using cAMP parameter assay kit. Proliferation was assessed using cell viability assay kit. RESULTS: The effects of hUCB-MSCs and modafinil on PAH are as follows. The right ventricular pressure (RVP) was reduced in the U and MD groups. The right ventricle (RV)/left ventricle (LV) + septum (S) ratio and RV/body weight (BW) were significantly decreased in the U and MD group. Decreased medial wall thickness of pulmonary arterioles and the number of intra-acinar arteries were also noted in both groups. Effects of hUCB-MSCs are as follows. The decreased mRNA and proteins expression levels of endothelin (ET)-1, endothelin receptor A, endothelial nitric oxide synthase and matrix metalloprotease-2 were observed by immunohistochemical staining in the U group. Inflammatory cytokines were decreased in the U group. As the effect of oral administration of modafinil, intermediate-conductance calcium-activated potassium channel (KCa3.1 channel), Bcl-2-associated X protein, vascular endothelial growth factor, tumor necrosis factor- α and interleukin-6 expression levels were significantly decreased in the lung tissues. Modafinil suppressed ET-1-induced PASMC proliferation via the KCa3.1 channel. CONCLUSION: hUCB-MSCs and modafinil improved hemodynamics and pulmonary pathologies in MCT-induced PAH rats. Therefore, these therapeutic approaches have a possibility of becoming new therapeutics for PAH.;폐동맥 고혈압 (PAH) 은 폐혈관 저항의 점진적인 증가로 인한 우심실 부전을 야기한다. 본 연구의 목적은 모노크로탈린으로 유도한 폐동맥 고혈압 백서 모델에서 사람 제대혈 유래 간엽 줄기세포 (hUCB-MSCs)와 모다피닐 (MD)의 효과를 규명하는 것이다. 다음과 같은 그룹으로 나누었다; 대조군 (C 군); 모노크로탈린으로 유도한 PAH 군 (M 군); PAH 유발 후 hUCB-MSCs 투여군 (U 군); PAH 유발 후 모다피닐 투여 군(MD 군). hUCB-MSCs (3 ×106 cells/rat) 투여는 모노크로탈린 투여 일주일 후 경정맥으로 하였다. MD 군은 세 개의 소군으로 나누었으며 경구투여하였다 (4, 10, 50 mg/kg/day). 먼저 hUCB-MSCs의 투여 효과는 다음과 같다. 평균 우심실압이 M군과 비교하였을 때 U 군에서 2주 째 유의하게 감소하였고, RV/(LV+S) 비율과 RV/체중 비율이 4주 째 유의하게 감소하였다. 폐세동맥 두께와 폐세동맥의 개수도 유의한 감소를 보였다. 엔토텔린-1 (ET-1), 내피세포유래 산화질소 합성효소 (eNOS), 금속 단백 분해효소-2 (MMP-2)의 mRNA 발현량이 4주 째 유의하게 감소하였으며, ET-1, 엔도텔린 수용체 A (ERA), eNOS, MMP-2의 단백질 발현량 감소가 관찰되었다. 염증 사이토카인 분비가 감소하였다. 두 번째로, 모다피닐의 효과는 다음과 같다. 평균 우심실압이 감소하고, 우심실 비대가 개선되었다. 병리학적 변화로서, 폐세동맥 중막 비후와 폐세동맥의 개수가 유의하게 감소하였다. ET-1, ERA, KCa3.1 채널의 단백질 발현량이 폐조직에서 유의하게 감소하였다. 이에 따라 폐동맥 고혈압이 개선된 결과 Bax, 혈관표피 성장인자, 종양괴사인자-α, 인터류킨-6의 발현량도 폐조직에서 유의한 감소를 보였다. 모다피닐은 KCa3.1 채널을 통해 ET-1으로 유도한 rPASMCs 증식식을 억제하였다. 폐동맥 고혈압 백서 모델에 hUCB-MSCs 또는 모다피닐을 투여 한 후, 평균 우심실압과 우심실 비대가 개선되었다. 병리학적 측면에서 개선이 있었으며 ET-1, ERA를 포함한 일부 단백질 발현 또는 mRNA발현이 개선되었다. 본 연구는 hUCB-MSCs의 경정맥 주입과 모다피닐의 경구투여가 PAH 개선 효과가 있음을 규명한 첫 연구라는 점에 의의가 있다.
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