Augmentation of Muscle Blood Flow by Ultrasound Cavitation Is Mediated by ATP and Purinergic Signaling.
BACKGROUND
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.


METHODS
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 2×108lipid 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 KATPchannels; 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.


RESULTS
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.


CONCLUSIONS
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.


BACKGROUND
URL: http://clinicaltrials.gov. Unique identifier: NCT01566890.
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.).
J Todd
JT
Belcik
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.).
Brian P
BP
Davidson
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.).
Aris
A
Xie
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.).
Melinda D
MD
Wu
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.).
Mrinal
M
Yadava
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.).
Yue
Y
Qi
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.).
Sherry
S
Liang
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.).
Chae Ryung
CR
Chon
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.).
Azzdine Y
AY
Ammi
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.).
Joshua
J
Field
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.).
Leanne
L
Harmann
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.).
William M
WM
Chilian
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.).
Joel
J
Linden
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.). lindnerj@ohsu.edu.
Jonathan R
JR
Lindner
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D006439:Hemodynamics; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D045423:Microbubbles; D018482:Muscle, Skeletal / Q000098:blood supply*; D058905:Purinergic Agents / Q000378:metabolism*; D015398:Signal Transduction; D014463:Ultrasonography / Q000379:methods*
D058905:Purinergic Agents; D000255:Adenosine Triphosphate
contrast echocardiography; microbubbles; microcirculation; perfusion