Belcik J Todd, Davidson Brian P, Xie Aris, Wu Melinda D, Yadava Mrinal, Qi Yue, Liang Sherry, Chon Chae Ryung, Ammi Azzdine Y, Field Joshua, Harmann Leanne, Chilian William M, Linden Joel, Lindner Jonathan R
From Knight Cardiovascular Institute (J.T.B., B.P.D., A.X., M.Y., Y.Q., S.L., C.R.C., A.Y.A., J.R.L.), and Oregon National Primate Research Center (J.R.L.), Oregon Health & Science University, Portland; Doernbecher Children's Hospital, Portland, OR; Division of Hematology and Oncology, Medical College of Wisconsin, Milwaukee (J.F., L.H.); Blood Center of Wisconsin, Madison, WI (J.F., L.H.); Northeast Ohio Medical University, Rootstown (W.M.C.); and Department of Pharmacology, Division of Development Immunology, La Jolla Institute for Allergy and Immunology, University of California San Diego (J.L.).
Circulation. 2017 Mar 28;135(13):1240-1252. doi: 10.1161/CIRCULATIONAHA.116.024826. Epub 2017 Feb 7.
Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signaling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation.
Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for 10 minutes after intravenous injection of 2×10 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or K channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease.
Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of endothelial nitric oxide synthase abolished the effects of therapeutic ultrasound, indicating downstream signaling through both nitric oxide and prostaglandins.
Therapeutic ultrasound using microbubble cavitation to increase muscle perfusion relies on shear-dependent increases in ATP, which can act through a diverse portfolio of purinergic signaling pathways. These events can reverse hindlimb ischemia in mice for >24 hours and increase muscle blood flow in patients with sickle cell disease.
URL: http://clinicaltrials.gov. Unique identifier: NCT01566890.
治疗性超声增强组织血流被认为依赖于对流剪切力。经历超声介导空化的微泡造影剂可显著放大这些效应。我们推测嘌呤能信号传导负责治疗性空化期间肌肉灌注的剪切力依赖性增加。
在静脉注射2×10脂质微泡后，对小鼠的近端后肢进行单侧暴露于治疗性超声（1.3MHz，机械指数1.3）10分钟（小鼠有或没有通过髂结扎产生的缺血）。通过低功率造影超声灌注成像评估微血管灌注。通过荧光素酶 - 荧光素酶测定法评估体内肌肉ATP释放以及内皮细胞或红细胞的体外ATP释放。通过研究以下干预措施评估嘌呤能信号传导途径：（1）加速ATP降解；（2）抑制P2Y受体、腺苷受体或钾通道；或（3）抑制涉及内皮型一氧化氮合酶或前列腺素产生的下游信号传导途径（吲哚美辛）。在一项针对12名稳定镰状细胞病患者的概念验证临床试验中评估超声空化对肌肉灌注的增强作用。
治疗性超声空化使正常小鼠的肌肉灌注增加7倍，在外周动脉疾病小鼠模型中使组织缺血逆转长达24小时，并使镰状细胞病患者的肌肉灌注增加一倍。血流增强远远超出超声暴露区域。超声空化使ATP产生约40倍的局部和持续增加，其来源包括内皮细胞和红细胞。抑制性研究表明，ATP是血流增强的关键介质，主要通过P2Y受体或外核苷酸酶活性产生的腺苷起作用。吲哚美辛与内皮型一氧化氮合酶抑制的联合作用消除了治疗性超声的作用，表明通过一氧化氮和前列腺素的下游信号传导。
使用微泡空化增加肌肉灌注的治疗性超声依赖于ATP的剪切力依赖性增加，其可通过多种嘌呤能信号传导途径起作用。这些事件可使小鼠后肢缺血逆转超过24小时，并增加镰状细胞病患者的肌肉血流量。
