Department of Pharmaceutical Sciences, College of Health Professions, North Dakota State University, Fargo, ND, USA.
Br J Pharmacol. 2019 Nov;176(22):4373-4387. doi: 10.1111/bph.14529. Epub 2018 Dec 9.
CD34 haematopoietic stem/progenitor cells have revascularization potential and are now being tested for the treatment of ischaemic vascular diseases in clinical trials. We tested the hypothesis that mitochondrial depolarization stimulates the reparative functions of CD34 cells.
Peripheral blood was obtained from healthy individuals (n = 63), and mononuclear cells (MNCs) were separated. MNCs were enriched for lineage negative cells, followed by isolation of CD34 cells. Vascular repair-relevant functions of CD34 cells, proliferation and migration, were evaluated in the presence and absence of diazoxide. Mitochondrial membrane potential, ROS and NO levels were evaluated by flow cytometry by using JC-1, mitoSOX and DAF-FM respectively.
Diazoxide stimulated the proliferation and migration of CD34 cells that were comparable to the responses induced by stromal-derived factor-1α (SDF) or VEGF. Effects of diazoxide were blocked by either 5-hydroxydecanoate (5HD), a selective mitochondrial ATP-sensitive potassium channel (mitoK ) inhibitor, or by L-NAME. Diazoxide induced mitochondrial depolarization, and NO and cGMP generation that were 5HD-sensitive. The generation of NO and cGMP by diazoxide was blocked by an endothelial NOS (eNOS)-selective inhibitor, NIO, but not by a neuronal (n)NOS-selective inhibitor, N -propyl-L-arginine (NPA). A Ca chelator, BAPTA, Akt inhibitor, triciribine, or PI3K inhibitor, LY294002, inhibited the NO release induced by diazoxide. Phosphorylation of eNOS at Ser and dephosphorylation at Thr were increased. Diazoxide-induced ROS generation and phosphorylation of eNOS at Ser were reduced by NPA.
Diazoxide stimulates vascular repair-relevant functions of CD34 cells via the mitoK -dependent release of NO and ROS.
This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc.
CD34 造血干/祖细胞具有血管生成潜力，目前正在临床试验中用于治疗缺血性血管疾病。我们验证了这样一个假设，即线粒体去极化刺激 CD34 细胞的修复功能。
从健康个体（n=63）中获取外周血，并分离单核细胞（MNC）。MNC 富集谱系阴性细胞，然后分离 CD34 细胞。通过使用 JC-1、mitoSOX 和 DAF-FM 分别评估 CD34 细胞的血管修复相关功能、增殖和迁移，在有无 diazoxide 的情况下进行评估。通过流式细胞术评估线粒体膜电位、ROS 和 NO 水平。
diazoxide 刺激 CD34 细胞的增殖和迁移，其作用与基质衍生因子-1α（SDF）或 VEGF 诱导的反应相当。5-羟基癸酸（5HD），一种选择性线粒体三磷酸腺苷敏感钾通道（mitoK）抑制剂，或 L-NAME 可阻断 diazoxide 的作用。diazoxide 诱导线粒体去极化和 NO 和 cGMP 的产生，5HD 敏感。内皮型一氧化氮合酶（eNOS）选择性抑制剂 NIO 可阻断 diazoxide 诱导的 NO 和 cGMP 的产生，但神经元（n）NOS 选择性抑制剂 N-丙基-L-精氨酸（NPA）不能阻断。钙螯合剂 BAPTA、Akt 抑制剂 triciribine 或 PI3K 抑制剂 LY294002 抑制 diazoxide 诱导的 NO 释放。eNOS 的 Ser 磷酸化和 Thr 去磷酸化增加。NPA 降低了 diazoxide 诱导的 ROS 生成和 eNOS 的 Ser 磷酸化。
diazoxide 通过 mitoK 依赖性 NO 和 ROS 的释放刺激 CD34 细胞的血管修复相关功能。
本文是关于线粒体药理学的主题部分的一部分：特色机制和治疗转化方法。要查看本节中的其他文章，请访问 http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc/。
