抑制血栓素及血栓素受體訊號減輕血管內皮素-1及缺血再灌流所引起的心臟損傷

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(1)國立臺灣師範大學生命科學系碩士論文. 抑制血栓素及血栓素受體訊號減輕血管內皮素-1 及缺血再灌流所引起的心臟損傷 Inhibition of Thromboxane A2/Thromboxane Receptor Signaling Attenuates Endothelin-1evoked and Ischemia/Reperfusion-induced Injury in Mouse heart. 研究生 : 丘幃尹 Wei-Yin Qiu. 指導教授 : 鄭劍廷博士 Chiang-Ting Chien, Ph.D.. 中華民國 106 年 08 月.

(2) 目錄 I. 中文摘要.................................................................................................................... 4 II. ABSTRACT .............................................................................................................. 6 III. ABBREVIATIONS.......................................................................................................... 8 IV. INTRODUCTION ................................................................................................... 10 1. THE CARDIOVASCULAR DISEASES IN WORLDWIDE...................................................... 10 2. MYOCARDIAL ISCHEMIA REPERFUSION INJURY ......................................................... 10 3. THE ASSOCIATION BETWEEN ENDOTHELIN-1 AND TXAS-TXA2-TP SIGNALING PATHWAY IN MYOCARDIAL ISCHEMIA REPERFUSION INJURY ......................................... 11. 4. USING TXAS OR TP GENE KNOCKOUT MICE FOR EXPLORING TXAS-TXA2-TP SIGNALING PATHWAY ..................................................................................................... 14. 5. AIM ........................................................................................................................... 14 V. MATERIALS AND METHODS .............................................................................. 15 1. ANIMALS ................................................................................................................... 15 1.1 WILD TYPE, TXAS AND TP GENE KNOCKOUT MICE. 15. 1.2 TXAS AND TP DOUBLE KNOCKOUT MICE:. 16. 1.3 SURGICAL PREPARATION. 16. 2. MEASUREMENT OF ELECTROCARDIOGRAPHIC PARAMETERS .................................... 17 3. CARDIAC MICROCIRCULATION DETERMINATION....................................................... 17 4. WIRE MYOGRAPHY .................................................................................................... 18 5. INDUCTION OF ACUTE MYOCARDIAL INFRACTION ..................................................... 19 6. IMMUNOHISTOCHEMISTRY (IHC) .............................................................................. 20 7. TERMINAL DEOXYNUCLEOTIDE TRANSFERASE DUTP NICK END LABELING STAIN ..... 21 8. MEASUREMENT OF CARDIAC TROPONIN I ................................................................... 23 9. STATISTICAL ANALYSES .............................................................................................. 23 VI. RESULTS ................................................................................................................ 24 1. HEART MICROCIRCULATION UNDER INTRAVENOUS NORMAL SALINE, U46619 AND ENDOTHELIN-1. ............................................................................................................. 24 2. ECG UNDER INTRAVENOUS NORMAL SALINE, U46619 AND ENDOTHELIN-1. ................ 25 3. EFFECT OF CONTRACTION AND RELAXATION IN MESENTERIC ARTERIES ..................... 26 4. MICROCIRCULATION AND ECG UNDER MYOCARDIAL ISCHEMIA REPERFUSION INJURY IN THREE GENOTYPE MICE. ............................................................................................ 27. 1.

(3) 5. HEMATOXYLIN AND EOSIN (H&E) STAIN .................................................................... 28 6. TERMINAL DEOXYNUCLEOTIDE TRANSFERASE DUTP NICK END LABELING STAIN ..... 28 7. IMMUNOHISTOCHEMISTRY ANALYSIS ......................................................................... 29 8. PLASMA LEVEL OF TROPONIN I MEASUREMENT. ......................................................... 30 VII. DISCUSSION ........................................................................................................ 31 1. PREVIOUS STUDIES OF TXAS-TXA2-TP SIGNALING AND ET-1 IN CARDIOVASCULAR DISEASES. ............................................................................... 31 2. IMPACT OF ECG AND HEART MICROCIRCULATION THROUGH TXAS-TXA2TP SIGNALING AND ET-1. ......................................................................................... 32 3. PHARMACOLOGICAL RESPOND IN RESISTANCE ARTERY THROUGH TXASTXA2-TP SIGNALING AND ET-1. ............................................................................... 33 4. INHIBITION OF TXAS-TXA2-TP SIGNALING ATTENUATE INJURY EVOKED BY MYOCARDIAL ISCHEMIA REPERFUSION THROUGH APOPTOSIS, OXIDATIVE STRESS, INFLAMMATION AND PYROPTOSIS. ................................ 34 VIII. CONCLUSION .................................................................................................... 39 IX. REFERENCE.......................................................................................................... 40 X. FIGURES AND TABLES ........................................................................................ 48 FIGURE. 1 ISCHEMIA/REPERFUSION MODEL IN MICE...................................................... 48 FIGURE. 2 CARDIAC MICROCIRCULATION UNDER INTRAVENOUS NORMAL SALINE IN A MINUTE. ........................................................................................................................ 49. FIGURE. 3 CARDIAC MICROCIRCULATION UNDER INTRAVENOUS U46619 (TP AGONIST, 2MG/KG) IN A MINUTE. ................................................................................................... 50 FIGURE. 4 CARDIAC MICROCIRCULATION UNDER INTRAVENOUS ET-1 (2.5 µG/KG) IN A MINUTE. ........................................................................................................................ 51. FIGURE. 5 CARDIAC MICROCIRCULATION UNDER INTRAVENOUS ET-1 (25 µG/KG) IN A MINUTE. ........................................................................................................................ 52. FIGURE. 6 CARDIAC MICROCIRCULATION UNDER INTRAVENOUS ET-1 (250 µG/KG) IN A MINUTE. ........................................................................................................................ 53. FIGURE. 7 (A) PERFUSION UNIT OF CARDIAC MICROCIRCULATION UNDER INTRAVENOUS NORMAL SALINE (RED ARROW) AMONG THREE GENOTYPE MICE. (B) THE PERCENTAGE OF PERFUSION UNIT AMONG MICE GENOTYPES.. .................................................................. 54. FIGURE. 8 (A) PERFUSION UNIT OF CARDIAC MICROCIRCULATION UNDER INTRAVENOUS U46619 (TP AGONIST, 2MG/KG, RED ARROW) AMONG THREE GENOTYPE MICE. (B) THE. 2.

(4) PERCENTAGE OF PERFUSION UNIT AMONG MICE GENOTYPES. ........................................ 55. FIGURE. 9 (A) PERFUSION UNIT OF CARDIAC MICROCIRCULATION UNDER INTRAVENOUS ET-1 (2.5µG/KG, 25 µG/KG AND 250µG/KG, RED, BLUE AND GREEN ARROW) AMONG THREE GENOTYPE MICE. ........................................................................................................... 56. FIGURE. 10 ECG OF THREE GENOTYPE MICE UNDER INTRAVENOUS NORMAL SALINE IN THREE MINUTES. ........................................................................................................... 57. FIGURE. 11 ECG OF THREE GENOTYPE MICE UNDER INTRAVENOUS U46619 (TP AGONIST, 2MG/KG) IN THREE MINUTES. ......................................................................................... 58 FIGURE. 12 FIGURE. 10 EKG OF THREE GENOTYPE MICE UNDER INTRAVENOUS ET-1(2.5, 25 AND 250 µG/KG). ........................................................................................................ 59 FIGURE. 13 THE R-R INTERVAL UNDER INTRAVENOUS (A) NORMAL SALINE, (B) U46619 (2MG/KG) AND (C) ET-1 (2.5, 25 AND 250 µG/KG) AMONG THREE GENOTYPE MICE. .......... 60 FIGURE. 14 EFFECT OF CONTRACTION AND RELAXATION IN MESENTERIC ARTERIES. (A) NOREPINEPHRINE (B) ACETYLCHOLINE (C) U46619 (D) ENDOTHELIN-1. ........................ 62 FIGURE. 15 CARDIAC MICROCIRCULATION (A) AND ECG (B) WITH MYOCARDIAL ISCHEMIA/REPERFUSION INJURY IN B6 MICE. ................................................................. 63. FIGURE. 16 CARDIAC MICROCIRCULATION (A) AND ECG (B) WITH MYOCARDIAL ISCHEMIA/REPERFUSION INJURY IN TXAS-/-TP+/+ MICE. ................................................. 64. FIGURE. 17 CARDIAC MICROCIRCULATION (A) AND ECG (B) WITH MYOCARDIAL ISCHEMIA/REPERFUSION INJURY IN TXAS-/-TP-/- MICE. .................................................. 65. FIGURE. 18 HISTOLOGICAL FEATURE OF HEART............................................................. 66 FIGURE. 19 TERMINAL DEOXYNUCLEOTIDE TRANSFERASE DUTP NICK END LABELING STAIN............................................................................................................................. 67. FIGURE. 20 IMMUNOHISTOCHEMISTRY OF BECLIN-1 ..................................................... 68 FIGURE. 21 IMMUNOHISTOCHEMISTRY OF IL-1Β ........................................................... 69 FIGURE. 22 IMMUNOHISTOCHEMISTRY OF 4-HNE ......................................................... 70 FIGURE. 23 PLASMA TROPONIN-I CONCENTRATION AFTER I/R. ..................................... 71. 3.

(5) I. 中文摘要心血管疾病包括冠心病、心絞痛、急性心肌梗塞等等，為世界死因之首。根據台灣衛生福利部的統計，我國心血管疾病為第二大死因，每十萬人就有 88.1 人於心血管疾病，僅次於惡性腫瘤的 199.6 人。心肌缺血會使心肌細胞發生強烈的發炎反應且再灌流後會使冠狀動脈釋放出大量活性氧物質，而活性氧物質又促進血栓的形成，影響到血栓素合成酶 (Thromboxane A2 synthase, TXAS)-血栓素 (Thromboxane A2, TXA2)- 血栓素受體. (Thromboxane prostanoid receptor, TP. receptor) 訊息傳遞路徑，包括增加了血栓素合成酶 (Thromboxane A2 synthase, TXAS) 的表達和血栓素受體 (Thromboxane prostanoid receptor, TP receptor)的活性，最終增加了血管內皮素(Endothelin-1, ET-1)釋放造成更加嚴重的傷害。本篇研究即是要探討抑制掉 TXAS-TXA2-TP 訊息傳遞路徑後，ET-1 所喚起的傷害和在心肌缺血再灌流的傷害上扮演著什麼樣的角色。我們使用三種不同基因型的老鼠， TXAS+/+TP+/+、TXAS-/-TP+/+及 TXAS-/-TP-/-小鼠並將實驗分為以 4.

(6) 下幾組。 1. 所有的小鼠將被隨機靜脈注射生理實驗水、 U46619 (TXA2 agonist, 2 mg/kg)和 ET-1(0.001-0.2 µg/kg)測量其心臟微循環，共四組(各組 N=6)。 2. 所有的小鼠將被隨機執行血管環模型實驗 (wire myography) ，測量其血管對藥物 (norepinephrine, acetylcholine, U46619, ET-1 )的收縮或舒張反應，共四組 (各組 N=6)。 3. 所有的小鼠將被隨機執行心肌缺血再灌流模型 (myocardial ischemia/reperfusion model)手術，共四組(各組 N=6)。並搭配組織免疫染色、血漿心肌旋轉蛋白(Troponin I)含量測定來看探討其細胞凋亡、細胞自噬、發炎性細胞凋亡和發炎之分生機轉及心肌梗塞之嚴重程度。我們的研究結果指出抑制 TXAS-TXA2-TP 訊息傳遞路徑，可以有效的減少心肌缺血再灌流所引起的細胞凋亡、發炎性細胞凋亡和氧化壓力，對小鼠具有較佳之心臟保護效果。. 關鍵字:缺血再灌流傷害、急性心肌梗塞、血栓素及血栓素受體訊息、血管內皮素-1. 5.

(7) II. Abstract Cardiovascular disease has become one of the most harmful human diseases with highest morbidity in the world, especially coronary heart disease and myocardial infarction. According to Ministry of Health and Welfare, the cardiovascular disease is the second cause of death and accounts for 0.881‰ death per year in Taiwan, second only to cancer. Myocardial ischemia/reperfusion (I/R) induces the release of oxidants in coronary arteries. Following the production, the oxidants may activate platelets and consequently induce thrombus formation. It is well known that thromboxane A2 synthase (TXAS) —thromboxane A2 (TXA2)—thromboxane prostanoid receptor (TP)— would activate TP, and increasing release of endothelin-1 (ET-1) to bring about more serious injury. In order to explore the role of TXAS-TXA2-TP pathway in endothelin-1 (ET-1) activation during I/R injury, we utilized mouse with gene depletion in TXAS (TXAS–/–) and both TXAS and TP (TXAS–/–TP–/–) mice. All mice were randomly subjected to intravenous normal saline, endothelin-1 (0.001-0.2 µg/kg body weight), U46619 (TXA2 agonist, 2 mg/kg body weight). Using Wire myograph model to determine the vascular reactivity of rat mesentery arteries, we investigated the possible signaling pathway between ET-1 and TXAS-TXA2-TP. In myocardial I/R model, the cardiac. injuries. were. evaluated 6. by. microcirculation,.

(8) electrocardiogram and plasma troponin I. We explored the mechanisms including apoptosis, pyroptosis and inflammation via level of plasma troponin I and immunohistochemistry stain in these animals. Our results indicate that the inhibition of TXAS-TXA2-TP pathway provides cardiac protection against myocardial I/R injury. Key words: Ischemia and Reperfusion Injury, Acute Myocardial. Infarction,. TXAS-TXA2-TP. Endothelin-1. 7. Signaling,.

(9) III. Abbreviations 4-HNE. 4-Hydroxynonenal. AA. Arachidonic acid. ACh. Acetylcholine. ASP. Aspirin. B6. B57CL/6J. BSA. Bovine serum albumin. CA. Coronary artery. CKD. Chronic kidney disease. COXs. Cyclooxygenases. CVD. Cardiovascular disease. ddH2O. Double-distilled water. ECG. Electrocardiography. ES cell. Embryonic stem cell. ET-1. Endothelin-1. ETA. Endothelin-1 type A receptor. H&E. Hematoxylin and eosin. H2O2. Hydrogen peroxide. HRP. Horseradish peroxidase. IHC. Immunohistochemistry. IL-1β. Interleukin-1 beta. IL-6. Interleukin-6. I/R injury. Ischemia/reperfusion injury 8.

(10) KH buffer. Krebs-Henseleit buffer. KO. Knockout. LAD. Left anterior descending. MA. Mesenteric artery. NE. Norepinephrine. NTNU. National Taiwan Normal University. PBST. Phosphate buffer saline tween-20. PE-10. Polyethylene 10. PE-60. Polyethylene 60. PGK. Phosphoglycerate kinase. PGK-HPRT. Phosphoglycerate kinase-hypoxanthine phosphoribosyltransferase. PGs. Prostaglandins. PU. Perfusion unit. ROS. Reactive oxygen species. SEM. Standard error of the mean. TXAS. Thromboxane A2 synthase. TP receptor. Thromboxane prostanoid receptor. TUNEL. Terminal deoxynucleotide transferase dUTP Nick End Labeling. TXA2. Thromboxane A2. U46619. 9,11-Dideoxy-11α,9αepoxymethanoprostaglandin F2α, TP receptor agonist. 9.

(11) IV. Introduction 1. The cardiovascular diseases in worldwide Global mortality rates from non-communicable diseases remain unacceptably high and are increasing. More than 70% of global cardiovascular disease (CVD) such as actual myocardial infraction, angina, hypertension and so on, are attributable to major risk factors (Ezzati, Majid, et al. 2003). 2. Myocardial ischemia reperfusion injury Myocardial ischemia reperfusion (I/R) injury occurs when the blood flow to the myocardium is interrupted, followed by the restoration of blood to the ischemic heart, for example, myocardial ischemia, cardiac surgery or circulatory arrest (Frank, Anja, et al. 2012). In general, I/R injury consists ischemic injury and reperfused injury. Ischemia to a specific region of the heart can evoke myocardium damage. According to previous studies, the source of ischemia injury is attribute to lower intracellular pH caused by lack of oxygen and mitochondrial dysfunction (Raedschelders, et al. 2012). The heart is particularly fragile because it needs high energy to maintain normal function. Early and fast restoration of blood flow to the heart has been established to be the treatment of choice to prevent further tissue injury (Raedschelders, et al. 2012). The use of thrombolytic therapy or primary percutaneous coronary intervention is the most effective strategy for decreasing the size of infarction in clinical. However, reperfusion injury is beginning upon restoration of blood flow to 10.

(12) the heart. This phenomenon can paradoxically reduce the beneficial effects of myocardial reperfusion. Numerous studies have observed the reduced cardiac function and even the acceleration of myocardial injury after reperfusion injury. Yellon (2007) show several major mediators of lethal reperfusion injury as following.. 3. The association between endothelin-1 and TXAS-TXA2-TP signaling pathway in myocardial ischemia reperfusion injury There are many metabolite products from arachidonic acid (AA) such as prostaglandins (PGs), leukotrienes. TXA2 is one of PGs generated through TXAS and it triggers multiple biological responses. via. its. cell. surface. TP-receptor. to. mediate. pathophysiologic conditions (Nakahata, 2008). It is not clear in the 11.

(13) relationship between thromboxane A2 (TXA2)- TXA2 synthase (TXAS)- thromboxane prostanoid (TP) receptor signaling and cardiovascular diseases. A wide distribution of TP receptors in different organs suggests that TXA2 is involved in a wide range of pathophysiological conditions. Nakahata showed the possible signaling pathways of TXAS-TXA2-TP in 2008.. The TP receptors and TP ligands, including TXA2, isoprostanes and prostaglandin H2, are elevated in vascular and atherothrombotic diseases (DeFilippis, Andrew, et al., 2013). It is well established that TXA2 is abnormally released during atherothrombotic diseases in humans (Fitzgerald, et al., 1986) and in animal models (Kuzuya and Tsunehiko, 1987), which mediates several pathophysiological states and events due to its biological activities as platelet aggregation, constriction of vascular smooth muscles, leukocyte chemotaxis and smooth muscle cells proliferation. Previous studies have shown an elevation of plasma 12.

(14) level of TXA2 after myocardial I/R injury in animal models (Sui, et al., 2004). However, the significance of TXAS-TXA2-TP signaling pathway in myocardial I/R injury is unknown. In addition to the contribution of TXAS-TXA2-TP signaling to myocardial I/R injury, endothelin-1 (ET-1) also plays a pivotal role on CVD induced pathophysiology. The ET-1 is known as powerful vascular contraction factor and acts through its ETA receptor. A number of studies have found that ETA receptor is widely distributed in many kinds of cells (Davenport and Anthony, 2016). Elevated plasma concentration of ET-1 can be detected in the coronary circulation both in human with ischemic heart diseases and in animal model with myocardial I/R injury (Filep, et al. 1994). Wang and their team (2015) showed the association between endothelin-1 and TXAS-TXA2-TP signaling pathway and chronic kidney disease (CKD).. 13.

(15) Although CKD is a progressive disease, most patients die from CVD before reaching end-stage renal disease (Santoro, et al., 2014). In addition, the relationship between ET-1 and TXASTXA2-TP signaling pathway in CVD is not fully known. 4. Using TXAS or TP gene knockout mice for exploring TXAS-TXA2TP signaling pathway In order to explore the role of TXAS-TXA2-TP signaling in myocardial I/R injury, we used two types of gene knocked out (KO) mice, TXAS-/-TP+/+ and TXAS-/-TP-/-. Those KO mice express normal physiology function but fail to generate TXA2 or lack of TP receptor in response to AA stimulation. These two groups of mice are able to maintain normal mean arterial blood pressure and protect against AA metabolites-induced shock and death (Yu, et al., 2004). 5. Aim The aim of the present work was to determine whether the blockage of TXAS-TXA2-TP signaling may prevent or decrease I/R injury in KO mice suffering from 30 min ischemia followed by 120 min of reperfusion. The hemodynamic, histopathological, biochemical, and aggregrometric parameters were thoroughly evaluated.. 14.

(16) V. Materials and methods 1. Animals. 1.1 Wild type, TXAS and TP gene knockout mice Male C57BL/6J (B6) mice weighing 30 to 35 g were purchased from BioLASCO Taiwan Co. Ltd. The Tbxas1 KO mice are generated by Dr. Shu-Wha Lin’s group. A targeting vector is designed to replace the distal portion exon 9 of the endogenous gene with a phosphoglycerate kinasehypoxanthine phosphoribosyltransferase (PGK-HPRT) cassette. This construct is electroporated into E14TG2a embryonic stem (ES) cells. Correct targeted ES cells are injected into B6 (i.e. wild type and TXAS+/+TP+/+) blastocysts. Chimeric mice are bred with B6 for 10 generations, the homozygous Tbxas1 KO mutant mice were obtained by intercrossing the heterozygous Tbxas1 KO mice. The Tbxa2r KO mice are generated by Dr. Thomas M. Coffman’s group (Thomas, Mannon et al., 1998) and are provided by Dr. Shu-Wha Lin’s group. A neomycin resistance gene driven by the phosphoglycerate kinase (PGK) promoter is inserted into a unique SfiI site near the proximal end of exon 2. This insertion targeting vector disrupts the coding sequence of the TP gene in the third transmembrane domain. This construct is electroporated into E14TG2a ES cells. Correctly targeted ES cells are injected into B6 blastocysts. Chimeric mice are bred with B6 for 10 generations, 15.

(17) the homozygous Tbxa2r KO mutant mice are obtained by intercrossing the heterozygous Tbxa2r KO mice. 1.2 TXAS and TP double knockout mice: Tbxas1 KO and Tbxa2r KO mouse are crossbred to generate Tbxas1-Tbxa2r double KO in the B6 background. Male mice including TXAS-/-TP+/+ and TXAS-/-TP-/- mice were provided from National Taiwan University core laboratory. All mice were housed at the Experimental Animal Center, National Taiwan Normal University (NTNU), at a constant temperature and with a consistent light cycle (light from 0600 h to 1800 h) with a temperature- and humidity-regulated environment (12°C, 505% RH). Food and tap water were provided ad libitum. All mice in the experiment and surgery were maintained according to the international guidelines for care and use of laboratory animals. Experimental procedures involving animals were approved by the Committee on Animal Research of NTNU and implemented under the guidelines of the Committee.. 1.3 Surgical preparation In this study, the animals were anesthetized with urethane (1500 mg/kg; Sigma, Missouri, USA) injected intraperitoneally (i.p.). The tracheas were exposed via a midline cervical incision and were intubated with polyethylene 60 (PE-60). Under urethane 16.

(18) anesthesia, the trachea of mice was intubated for artificial ventilation (Small Animal Ventilator Model 683, Harvard Apparatus, Holliston, Mass), with 150 breaths in 1 minute, a tidal volume of 10 mL per kg, and a positive end-expiratory pressure of 2 cm H2O. The body temperature was monitored with a rectal thermometer and was kept at 36.5-37.0°C by an infrared light. 2. Measurement of Electrocardiographic Parameters Three-lead ECGs were measured using surface electrodes attached to the right arm, left arm, and left leg limb. The heart rate, heart rhythm, and wave forms were recorded using an iWorx 214 data recorder (IX-214; iWorx Systems, Inc, Dover, NH). 3. Cardiac Microcirculation Determination A full-field laser perfusion imager (MoorFLPI, Moor Instruments, Ltd, Devon, UK) was used to continuously quantify the microcirculatory blood flow intensity. The contrast imaging was used by laser speckle, which exploits the random speckle pattern generated when tissue is illuminated by laser light. The random speckle pattern changes when blood cells move within the region of interest. When a high level of movement (fast flow) is present, the changing pattern will become more blurred, and the contrast in that region will be accordingly reduced. The contrast image is processed to produce a 16-bit color-coded image that correlates with the blood flow in the heart, such that blue is defined 17.

(19) as low flow and red as high flow. The microcirculatory blood flow intensity of each region of interest was recorded as flux with the perfusion unit, which is related to the product of average speed and concentration of moving red blood cells in the heart sample volume. The negative control value was set at 0 perfusion unit (blue) and the positive value at 1000 perfusion units (red). The perfusion units were analyzed in real time using MoorFLPI software, version 3.0 (Moor Instruments, Ltd, Devon, UK) (Chien, et al., 2015) In this study, we used normal saline, ET-1 and U46619 intravenously injected to the mice through the jugular vein to investigate microcirculation of those in heart. Total mice were divided into following groups: normal group was given normal saline (B6, n = 3; TXAS-/-TP+/+, n = 3; TXAS-/-TP-/-, n = 3); ET-1 group was given different doseage of ET-1 (0.001-0.2 µg/kg body weight, B6, n = 3; TXAS-/-TP+/+, n = 3; TXAS-/-TP-/-, n = 3); U46619 group was given different dose of U46619 (TXA2 agonist, 2 mg/kg body weight, B6, n = 5; TXAS-/-TP+/+, n = 4; TXAS-/-TP-/-, n = 5). 4. Wire myography Wire myography on mouse mesenteric artery (MA) was performed as described elsewhere (Broegger, Matchkov, et al., 2011). In brief, after anesthesia (3% isoflurane and 100% oxygen), second order MA of 8-12 week old mice were carefully cleaned perivascular adipose tissue and dissected in Krebs-Henseleit (KH) buffer (118 mmol/L NaCl, 3.4 mmol/L KCl, 1.2 mmol/L KH2PO4, 18.

(20) 1.2 mmol/L MgSO4, 25 mmol/L NaHCO3, 11 mmol/L glucose, 1 mmol/L CaCl2, bubbled with 95% O2/5% CO2 at pH=7.4) and cut into 2 mm segments. The vessel rings were then carefully mounted in a wire myography (620M, Danish MyoTechnology, Aarhus, Denmark) using two stainless steel wires (d = 25 μm) for MAs. The vessels were let to equilibrate for 60 min at 37oC, and then gradually stretched up to 20 mN for MA. After normalization, vessels were left to equilibrate for another 30 min, and tested for viability using norepinephrine (NE) and acetylcholine (ACh). Contractions to ET-1 (10-12 to 10-8 mol/L), NE (10-9 to 10-3 mol/L), and U46619 (10-9 to 10-5 mol/L) and relaxation to ACh (10-9 to 103. mol/L) were determined.. 5. Induction of Acute Myocardial Infraction Mice were subjected to a myocardial ischemia and reperfusion model as described (Oyama, Jun-ichi, et al., 2004; Chien, et al., 2014). After surgical preparation, the chest was opened by a horizontal incision through the skin and muscle layers. An incision was made in the muscle between the ribs, and they were separated with a retractor to expose the heart. Ischemia was achieved by ligating the anterior descending branch of the left coronary artery (LAD) by using a 7-0 silk suture with a 1-mm section of PE-10 tubing placed on top of the LAD, 2 to 3 mm from the tip of the normally positioned left atrium. Regional ischemia was confirmed by visual inspection of pale color in the occluded 19.

(21) distal myocardium. After occlusion for 30 minute, reperfusion occurred by releasing the ligature and removing the PE-10 tube (Figure 1). This allowed reperfusion of the formerly ischemic area. Blood flow was confirmed by visualization of the return of a bright red color in the previously pale region. After 2 hours reperfusion, the animals were sacrificed with an intravenous KCl injection (0.1 mg/mL). The heart was immediately removed and divided into two parts. One part was stored in 10% neutral buffered formalin for immunohistochemistry assay, and another was frozen in liquid nitrogen and stored at –80◦C for protein isolation or other assay. 6. Immunohistochemistry (IHC) Tissue sections were deparaffinized in xylene and rehydrated in ethanol. The slides were immersed in xylene for 10 minutes at room temperature and repeated using fresh xylene for second 10 minutes incubation. The slides were sequentially immersed in 100%, 95%, 75% and 50% ethanol each for 5 minutes at room temperature. Then the tissue sections were submitted to antigen retrieval buffer for 20 minutes at 95oC. The retrieval buffer solution used for heat-induced epitope retrieval was sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0). After 20 minutes, we applied 3% hydrogen peroxide (H2O2) to clean H2O2 enzyme for 15 minutes at room temperature and then the slides were blocked for non-specific binding with 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at 20.

(22) room temperature and incubated with the primary antibodies for 18 hours at 4°C. Primary antibodies included rabbit anti 4hydroxynonenal (4-HNE, 1:500; Alpha Diagnostic, San Antonio, TX, USA) and mouse anti Interleukin-1 beta (IL-1β, 1:1000; Cell Signaling Technology, Denver, MA, USA). Tissue sections were washed with phosphate buffer saline tween-20 (PBST) three times and then incubated with secondary antibodies HRP-conjugated rabbit anti-mouse immunoglobulin G (IgG) or goat anti-rabbit IgG (1:800; both from Sigma Aldrich St. Louis, MO, USA) for 1 hour at room temperature. After washing with PBST for 3 times, we immersed slides in DAB (ImmPACT DAB Peroxidase Substrate; Vector, Burlingame, California, USA) for 3-5 minutes, washed with double-distilled water (ddH2O), and immersed slides in hematoxylene for 5 minutes. The slides were dehydrated in ethanol series and were mounted in mounting medium (Leica, Wetzlar, Germany). 7. Terminal deoxynucleotide transferase dUTP Nick End Labeling We used BioVision’s Apo-BrdU-IHCTM Kit to performed a two-color TUNEL stain (Terminal deoxynucleotide transferase dUTP Nick End Labeling) (catalog #K403-50; 50 assays; stored at -20oC) assay for labeling DNA breaks to detect apoptotic cells by immunohistochemistry. The slides were immersed in xylene for 10 minutes at room temperature and repeated using fresh xylene for another 10 minutes incubation. The slides were sequentially immersed in 100%, 95%, 75% and 50% ethanol each for 5 minutes 21.

(23) at room temperature. We immersed slides into 1X PBST, dried the glass slide and applied 100 µl Proteinase K solution at 1:100 in 10 mM Tris with pH 8 of entire specimen for 20 minutes at room temperature. The slides were then rinsed with 1X PBST, gently tapped off excess liquid and applied 100 µl of 3% H2O2 at 30% H2O2 1:10 in methanol for 5 minutes at room temperature. We rinsed the slide with 1X PBST, gently tapped off excess liquid and carefully dried the glass slide then applied entire specimen with 1X Reaction Buffer at 5X diluted Reaction Buffer in ddH2O for 10 to 30 minutes at room temperature. We carefully blotted the 1X Reaction Buffer from the specimen and immediately applied 50 µl of Complete Labeling Reaction Mixture onto each specimen, which were then covered with a piece of Parafilm for 1 to 1.5 hours at 37°C. After that, we removed Parafilm cover slip, rinsed slide with 1X PBST, gently tapped off excess liquid and carefully dried the glass, then applied 100 µl of Blocking Buffer for 10 minutes at room temperature. We immediately covered specimen with 100 µl of Antibody Solution in the dark for 1-1.5 hours at room temperature after blotted the Blocking Buffer from the specimen. We washed the specimen with 1X PBST and covered the entire specimen with 100 µl of Blocking Buffer and 100 µl of conjugate at 200X Conjugate 1:200 in Blocking Buffer for 30 minutes at room temperature. Five minutes before concluding incubation, we prepared DAB solution by dissolving one tablet of DAB and one 22.

(24) tablet of H2O2/Urea in 1 ml of tap H2O and the specimen were tapped off excess liquid, covered with 100 µl of DAB solution for 15 minutes at room temperature. We rinsed slides with ddH2O and immediately covered the entire specimen with 100 µl of Methyl Green Counterstain solution for 2 to 3 minutes at room temperature. At the end, the slides were dehydrated in ethanol series and were mounted in mounting medium. 8. Measurement of cardiac troponin I Cardiac troponin I levels, a second measure of cardiac injury, were determined by Taipei City Hospital Ren-Ai Branch. Blood was obtained in the time of sacrifice and then plasma was separated by ultracentrifuge (14000 rpm, 30 minutes). Plasma was prepared from each test group and stored at − 80°C. 9. Statistical analyses Data are reported as mean ± standard error mean (SEM). The statistical difference was considered significant when P < 0.05. We analyzed the data with two-sided unpaired Student’s t-test. We performed all analyses with Prism3 (GraphPad; San Diego, CA).. 23.

(25) VI. Results 1. Cardiac microcirculation under intravenous normal saline, U46619 and Endothelin-1. The direct image measurement of change of cardiac microcirculation within three minutes was shown in Figure 2~6, representing the normal saline, U46619 and ET-1 group respectively. According to Figure 2, no significant change of cardiac microcirculation was found in B6, TXAS-/-TP+/+ and TXAS-/-TP-/- mice under intravenous normal saline. According to Figure 3, a marked decrease of cardiac microcirculation was found in B6, TXAS-/-TP+/+ but TXAS-/-TP-/- mice under intravenous U46619 within 30 sec-150 sec. However, the decrease of cardiac microcirculation recovered immediately within three minutes. Figure 4~6 showed a dose-dependent decrease of cardiac microcirculation in B6, TXAS-/-TP+/+ and TXAS-/-TP-/- mice under intravenous ET-1. According to Figure 4, no marked change of cardiac microcirculation was found in B6, TXAS-/-TP+/+ and TXAS-/-TP-/- mice under intravenous low-dose ET-1 within three minutes. According to Figure 5, a slight change of cardiac microcirculation was found in B6 but TXAS-/-TP+/+ and TXAS-/TP-/- mice under intravenous ET-1 within 30 sec-50 sec. The decrease of cardiac microcirculation recovered within three minutes. According to Figure 6, an obvious change of cardiac microcirculation was found in B6 but TXAS-/-TP+/+ and TXAS-/TP-/- mice under intravenous ET-1 within 30 sec-50 sec. The 24.

(26) reduction of cardiac microcirculation recovered immedilately within three minutes. The continuous time recording of perfusion unit (PU) under normal saline, U46619 and ET-1 group were demonstrated in Figures 7-9. The comparison of PU among B6, TXAS-/-TP+/+ and TXAS-/-TP-/- mice under intravenous normal saline, U46619 and ET-1 were shown in Figures 7-9. There is no significant change of PU among B6, TXAS-/-TP+/+ and TXAS-/-TP-/- mice under intravenous normal saline. A significant decrease of PU between B6 and TXAS-/-TP+/+ mice compared with TXAS-/-TP-/- (P<0.05) within 30S~150S. Decreasing of PU was significantly found in B6 compared with TXAS-/-TP+/+ and TXAS-/-TP-/- mice under middle and high dose ET-1 within 20S~50S (P<0.05). 2. ECG under intravenous normal saline, U46619 and Endothelin-1. The change of ECG in B6, TXAS-/-TP+/+ and TXAS-/-TP-/mice under intravenous normal saline, U46619 and ET-1 are shown in Figures 10-12. When TXAS-/-TP+/+ and TXAS-/-TP-/mice were suffered from intravenous normal saline (0 min), no change of ECG was found compared with their baseline (-1 min) within three minutes in Figure 10. U46619 was intravenously injected into B6 and TXAS-/-TP+/+ mice, bradycardia or arrhythmia was observed within three minutes in Figure 11. After about three minutes, TXAS-/-TP+/+ mice recover but B6 need more time to recover to baseline. TXAS-/-TP-/- mice stay baseline when use U46619. ET-1 was intravenously injected into B6 mice, 25.

(27) bradycardia or arrhythmia was observed in high dose in Figure 12. TXAS-/-TP+/+ and TXAS-/-TP-/- mice stayed baseline when use ET1. The comparison of R-R interval among B6, TXAS-/-TP+/+ and TXAS-/-TP-/- mice under intravenous normal saline, U46619 and ET-1 were shown in Figure 13. No change of R-R interval when B6, TXAS-/-TP+/+ and TXAS-/-TP-/- mice were suffered from intravenous injection of normal saline compared with their baseline in Figure 13-a. Injection of U46619 have significant increasing of R-R interval in B6 and TXAS-/-TP+/+ mice was observed in Figure 13-b(P<0.05). When in one minute, TXAS-/TP+/+ express higher R-R interval than B6 but have no significance. When in two minutes, B6 express higher R-R interval than TXAS/-. TP+/+ but have no significance. R-R interval in TXAS-/-TP-/- mice. have no significance compared their baseline and lower than B6 when use U46619. Injection of ET-1 had no significant increasing of R-R interval in low and middle dose among three genotype mice. Injection of high dose ET-1 was found significant increasing of R-R interval in B6 and TXAS-/-TP+/+ mice in Figure 13c(P<0.05). B6 expressed higher R-R interval than TXAS-/-TP+/+ and TXAS-/-TP-/- mice. 3. Effect of contraction and relaxation in mesenteric arteries The effect of contraction and relaxation in MAs among B6, B6+aspirin (ASP), TXAS-/-TP+/+ and TXAS-/-TP-/- mice under NE, ACh, U46619 and ET-1. MAs from TXAS-/-TP+/+ and TXAS-/-TP/-. mice had significantly reduced contraction to NE, comparing 26.

(28) with B6 (P<0.05) in Figure 14-a. ASP havd no impact on contraction to NE. MAs from B6+ASP, TXAS-/-TP+/+ and TXAS-/TP-/- mice havd significantly sensitive relaxation to ACh (10-8~107. M, P<0.05), comparing with B6. In addition, MAs from B6+ASP,. TXAS-/-TP+/+ and TXAS-/-TP-/- mice maintain higher tension, comparing with B6 under high concentration of ACh in Figure 14-b. MAs from B6+ASP and TXAS-/-TP-/- mice had significantly reduced contraction to U46619, comparing with B6 and TXAS-/TP+/+ (P<0.05) in Figure 14-c. Although MAs from TXAS-/-TP+/+ mice show lower contraction to U46619 than B6, it had no significance. MAs from B6+ASP and TXAS-/-TP-/- mice havd significantly reduced contraction to ET-1, comparing with B6 and TXAS-/-TP+/+ (P<0.05) in Figure 14-d. Although MAs from TXAS-/-TP+/+ mice showed lower contraction to ET-1 than B6, it had no significance. 4. Cardiac microcirculation and ECG under myocardial ischemia reperfusion injury among three genotype mice The cardiac microcirculation of myocardial I/R injury among three genotype mice was determined and shown in Figure a of 15~17. All genotype mice suffered from ischemia operation express decreasing of blood flow (black hollow square) which LAD was ligated. When origin of reperfusion, cardiac microcirculation of all genotype mice was recovery. Figure b of 15~17 showed that S-T segment elevation appear in all genotype mice under ischemia and it didn’t recover under reperfusion. 27.

(29) 5. Hematoxylin and eosin (H&E) stain In order to examine the tissue abnormality, the histological heart sections were stained with H&E. The histological stain of heart was shown in Figure 18-a. I/R injury increased the intracellular split (edema), red blood cell extravasation, loss of cross striations in three genotype mice. According to Figure 18-b, there were the differences of degree of split formation could be compared among three genotype mice. All genotype mice showed significantly higher ratio of split formation than their control group (P<0.05). The significantly higher ratio of split formation in B6 I/R treated group was shown than TXAS-/-TP+/+ and TXAS-/-TP-/- I/R treated group (P<0.05). But it had no significance between TXAS-/-TP+/+ and TXAS-/-TP-/- I/R treated group. 6. Terminal deoxynucleotide transferase dUTP Nick End Labeling stain We used TUNEL stain to quantify the apoptotic cells formation and the DNA fragmentation occurred in the nucleus appeared brown color, which was shown in Figure 19-a. There are the differences of degree of apoptotic cell could be compared among three genotype mice in Figure 19-b. All genotype mice showed significantly higher ratio of apoptotic cell than their control group (P<0.05). The significantly higher ratio of apoptotic cell in B6 I/R treated group was shown than TXAS-/-TP+/+ and TXAS-/-TP-/- I/R treated group (P<0.05). But it had no significance between TXAS/-. TP+/+ and TXAS-/-TP-/- I/R treated group. 28.

(30) 7. Immunohistochemistry analysis To. further. confirm. the. possible. pathophysiologic. mechanisms of myocardial I/R injury, several IHC stains were performed. Tissue sections were stained to determine the degree of oxidative stress, inflammatory cytokines and autophagy in response to I/R injury. As shown in Figure 20-a, Beclin-1 is a marker for autophagy. There are significant differences of Beclin-1 expression could be compared among three genotype mice in Figure 20-b. All genotype mice showed significantly higher ratio of Beclin-1 than their control group (P < 0.05). The significantly higher ratio of Beclin-1 in B6 I/R treated group was shown than TXAS-/-TP+/+ and TXAS-/-TP-/- I/R treated group (P<0.05). But it had no significance between TXAS-/-TP+/+ and TXAS-/-TP-/- I/R treated group. IL-1β is a proinflammatory cytokines. It is a marker for examining pyrotosis, shown in Figure 21-a. There are the differences of degree of IL-1β could be compared among three genotype mice in Figure 21-b. All genotype mice showed significantly higher ratio of IL-1β than their control group (P<0.05). The significantly higher ratio of IL-1β in B6 I/R treated group was shown than TXAS-/-TP+/+ and TXAS-/-TP-/- I/R treated group (P<0.05). But it had no significance between TXAS-/-TP+/+ and TXAS-/-TP-/- I/R treated group. Oxidative injury represented by 4-HNE, it is a product of lipid peroxidation—is shown in Figure 22-a. There are the differences 29.

(31) of degree of 4-HNE could be compared among three genotype mice in Figure 22-b. All genotype mice showed significantly higher ratio of 4-HNE than their control group (P<0.05). The significantly higher ratio of 4-HNE in B6 I/R treated group was shown than TXAS-/-TP+/+ and TXAS-/-TP-/- I/R treated group (P<0.05). But it had no significance between TXAS-/-TP+/+ and TXAS-/-TP-/- I/R treated group.. 8. Plasma level of troponin I measurement. Troponin I level is an indirect marker to evaluate the infraction size. Troponin I concentration was determined in three genotype mice under myocardial I/R injury in Figure 23. The Troponin I level were significantly elevated in all I/R mice compared with their sham control group (B6, P < 0.001; TXAS-/TP+/+ and TXAS-/-TP-/-, P < 0.05). I/R treated B6 had significantly higher troponin I concentration than IR treated TXAS-/-TP+/+ and TXAS-/-TP-/- mice (P < 0.01). However, there is no significance in troponin I level between IR treated TXAS-/-TP+/+ and TXAS-/-TP-/mice.. 30.

(32) VII. Discussion 1. Previous studies of TXAS-TXA2-TP signaling and ET-1 in cardiovascular diseases. TXAS-TXA2-TP signaling paly important bioactivities. TXAS-/-TP+/+ mice cannot biosynthesize TXA2 because there is no TXAS gene expression. There are no TXAS and TP gene expression in TXAS-/-TP-/- mice, it is not only TXA2 cannot be produced but also TP receptor cannot be presented. Especially, it is not only one ligand can trigger TP receptor to reach downstream respond. Precious studies about TXAS-TXA2-TP signaling usually focused on thrombotic heart diseases such as anti-atherosclerosis, anti-platelet aggregation. less studies in acute myocardial infarction. In addition, ET-1 is the strongest known vascular contraction factor (Yanagisawa, et al., 1988). Several cardiovascular pathologies are associated with increasing level of circulating ET-1, such as congestive heart failure, pulmonary hypertension, coronary artery disease, ischemic heart disease and acute myocardial infarction (McMurray, et al., 1992; Giaid, et al., 1993). There has been an elevation in the concentration of ET-1 in the plasma of patients with myocardial infarction and Setianto and their team have reported currently that the increasing level of plasma ET-1 in S-T segment elevation acute myocardial infarction (STEMI). However, report of the relation between TXAS-TXA2-TP signaling and ET-1 is rare. 31.

(33) Our experiments showed the change of heart microcirculation through TXAS-TXA2-TP signaling first time. 2. Impact of ECG and cardiac microcirculation through TXASTXA2-TP signaling and ET-1. An extreme reduction of cardiac microcirculation was observed when U46619 was injected intravenously into B6 and TXAS-/-TP+/+ mice but TXAS-/-TP-/- mice. As a powerful vasocontraction factor, a strong contractile response was generated via TXAS-TXA2-TP signaling (Shen, et al., 1998). In the experiment of intravenous injection different dose of ET-1 (low, middle, high). Our data showed that reduction of cardiac microcirculation in middle and high dose of ET-1 in B6 mice but TXAS-/-TP+/+ and TXAS-/-TP-/- mice. As the strongest vasocontraction factor, most of the myocardium and vessels have ETA receptor induced a contractile response. Wang and their team has reported that over activation of TXAS-TXA2-TP signaling lead to upregulation of ETA receptor with CKD mice. The reason of the poor respond to ET-1 in TXAS-/-TP+/+ and TXAS-/-TP-/- mice, we speculated that decrease the expression of ETA receptor via inhibition of TXAS-TXA2-TP signaling. We also observed that bradycardia or arrhythmia appear when U46619 was injected into B6 and TXAS-/-TP+/+ mice but TXAS-/TP-/- mice. S-T segment elevation had been reported in rats with intracoronary injection of U46619, while the study didn’t 32.

(34) indicate that U46619 result in bradycardia or arrhythmia (Yamamoto, et al., 1993). Dosage of U46619 (10ug/kg in rat vs 2mg/kg in mice) and location (coronary artery vs jugular vein) are different from our research. In addition to respond caused by U46619, intravenous injection of ET-1 (high dose) has a similar phenomenon. 3. Pharmacological respond in resistance artery through TXASTXA2-TP signaling and ET-1. Due to intravenous injection take the drug to the whole body, so we cannot ensure those respond from coronary arteries or myocardium. Moreover, we perform contraction effect to NE, U46619 and ET-1, relaxation effect to ACh via MAs. There are two reasons why we choose the MA to simulate the coronary artery (CA). One of two is MA and CA belong to resistance artery and the diameter of the MA we used is equal to the diameter of the CA (about 200 um). Another reason is that right gastroepiploic artery (GEA) has been used to complete clinical coronary artery bypass graft. The GEA graft is a safe and effective arterial conduit for coronary artery bypass grafting (Suma, et al., 2013). According to this, the arteries of the digestive system can be used as a CA. Reduction of vessels tension to NE in TXAS-/-TP+/+ and TXAS-/-TP-/- mice indicate that inhibition of TXAS-TXA2-TP signaling maybe decrease sympathetic activity via decreasing adrenergic receptors (α1 receptor). Relaxation of vessels tension to low concentration 33.

(35) ACh (10-9~10-7M) in B6+ASP, TXAS-/-TP+/+ and TXAS-/-TP-/mice are more sensitive than B6 mice. The strongest relaxation of vessels tension to high concentration ACh (10-5~10-3M) was reached in B6 mice. B6 mice presents a wide range of vessels tone than those who inhibition of TXAS-TXA2-TP signaling mice. This result we explain in the face of sudden changes in blood pressure such as septic shock still possessing a better function to maintain homeostasis in individuals with inhibition of TXAS-TXA2-TP signaling. Reduction of contraction of vessels tension to U46619 in B6+ASP and TXAS-/-TP-/- mice compared with B6 and TXAS-/-TP+/+ mice. Although vessels tension in TXAS-/-TP+/+ mice lower than B6, there is no significance between them. The data can be associated with the data of heart microcirculation. Reduction of contraction of vessels tension to ET-1 in B6+ASP and TXAS-/-TP-/- mice than B6 and TXAS-/TP+/+ mice. And when the concentration of ET-1 is 10-8M, vessels in TXAS-/-TP+/+ mice significantly present lower tension than B6. This result indicates that vessels contraction are induced via Et-1 are decreased through inhibition of TXAS-TXA2-TP signaling. According to this data, we confirm our hypothesis that TXAS-TXA2-TP signaling is upstream of ET-1 in cardiovascular function. 4. Inhibition of TXAS-TXA2-TP signaling attenuate injury evoked by myocardial ischemia reperfusion through apoptosis, oxidative. 34.

(36) stress, inflammation and pyroptosis. For many years, it was thought that myocardial reperfusion is only beneficial and that there was no cell death related to it (Braunwald, et al., 1985; Kloner, et al.,1993). Later when cardiomyocytes death was seen in the reperfusion myocardium it was postulated that they are the already irreversibly damaged cardiomyocytes that were fated to die during ischemia (Gottlieb, et al., 1994). The concept of ‘reperfusion injury was presented when it was shown that reperfusion induced death in cardiomyocytes that were viable during ischemia. The burst production of reactive oxygen species (ROS) during I/R stage might impair the function and structure of the tissue or organ by triggering several abnormal signal transductions to induce several types of cell death such as apoptosis, autophagy, pyroptosis, and necrosis (Chien CT, et al., 2012). Prostanoid, including prostaglandins (PGs) and thromboxane, are generated from AA by the enzyme cyclooxygenases (COXs). The role of PGs and its mechanism in apoptotic impairment with myocardial I/R injury had been reported (Qiu, Hong, et al., 2012). However, less studies discuss the role of TXA2 with myocardial I/R injury (Mullane, et al., 1988; Nichols, et al., 1989). We perform myocardial I/R model in three genotype mice to clarify whether suppress TXAS-TXA2-TP signaling could reduce I/R injury in mouse heart. We cannot find any difference on heart microcirculation in this model among three genotype mice. 35.

(37) Theoretically, the level of thrombosis and vasocontraction are decline via inhibition of TXAS-TXA2-TP signaling. But in this model, LAD was ligated physically so the situation in ischemia should be similar no matter what kind of animal. Therefore, we must to discuss the role of inhibiting TXA2-TP- signaling in myocardial I/R injury from other aspects such as HE stain, IHC stain, TUNEL and plasma troponin I. Represent views of heart section in B6 mice showing I/R treated heart with higher levels of intracellular split (edema), red blood cell extravasation and loss of cross striations. According to histological evidences, I/R injury on cardiomyocytes were attenuated through the inhibition of TXA2-TP- signaling. The increasing level of plasma IL-6 and neutrophil polymorphs infiltration was observed by HE stain had been reported on acute myocardial infarction (Hashmi, et al., 2015). But we didn’t find neutrophil polymorphs infiltration on our HE stain. Experimental studies provide strong but somewhat conflicting evidence that neutrophils are involved in the myocardial response leading to lethal injury upon reperfusion. Some anti-neutrophil interventions successfully reducing lethal reperfusion injury reported by some laboratories have not been reproduced by other laboratories using different or even similar animal models (Vinten-Johansen, et al., 2004). Although neutrophil polymorphs infiltration didn’t find in our HE stain data, IL-1β present in our IHC stain showed lower level through blocking TXA2-TP36.

(38) signaling. IL-1β is one of inflammation and pyroptosis markers. Pyroptosis is characterized by rapid plasma membrane rupture and release of proinflammatory intracellular contents, which is morphologically and mechanistically distinct from other forms of cell death (Yang, et al., 2014). Recently, a review demonstrated the main role of pyroptosis in I/R injury (Bell, et al., 2016). IL1β played a pivotal role in inflammation in myocardial I/R injury and vascular endothelial dysfunction (Nowak, et al., 2016). IL-1β was upregulated of pro- IL-1β through NF-κB mediated transcriptional activation and it activated TXAS and TP receptors through an auto-activation mechanism (Huang, et al., 2013). TUNEL stain show higher anti-apoptotic activity through the inhibition of TXA2-TP- signaling. It was shown by Lieberthal that the severity and duration of ATP depletion determines the mechanism of death: cells with an intracellular ATP concentration below a certain threshold become necrotic, whereas an ATP value above that threshold induces apoptosis (Shiraish, et al., 2001). As ischemia is associated with more ATP depletion, whereas reperfusion may replenish the ATP stores, the main mechanism of cell death is caspase activated apoptosis in ischemia reperfusion model. We also demonstrated that oxidative stress was attenuated in myocardial I/R injury via 4-HNE through inhibition of TXA2TP- signaling. Main impairment of reperfusion result from oxidative stress had been discussed a long time. Various studies 37.

(39) have demonstrated that generation of ROS in I/R injury induced injury or oxygen-derived free radicals can lead to programmed cell death (Zweier, et al., 1988; Buttke, et al., 1994). Unlike most of using evans blue&TTC double stain to identify the myocardial infarct size (Price, et al., 2011), we have utilized troponin I to examine the degree of infarction (Mair, et al., 1995; Hallén, et al., 2009). Our data indicate it have better cardioprotection when blocking TXA2-TP- signaling.. 38.

(40) VIII. Conclusion In summary, this study can divide into two parts. (1) In order to elaborate the relationship between TXA2-TP- signaling and ET1 on heart microcirculation and function. Inhibition of TXA2-TPsignaling gives a cardioprotection when faced with challenges from ET-1 and TXA2 and substantiates that TXA2-TP-signaling is located upstream of ET-1 via pharmacological experiments. (2) In order to evaluate whether injury evoked by I/R was attenuated with blocking TXA2-TP- signaling. I/R model was performed to explain reducing of apoptosis, oxidative, inflammation, pyroptosis and degree of infarction. This research will benefit the development of new therapeutic strategies, or to ameliorate the old treatments in the future.. 39.

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(49) X.. Figures and Tables. Figure. 1 Myocardial ischemia/reperfusion model in mice. Ischemia is achieved by ligating LAD by using a 7-0 silk suture with a 1mm section of PE-10 tubing placed on top of the LAD, 2 to 3 mm from the tip of the normally positioned left atrium. Regional ischemia is confirmed by visual inspection of pale color in the occluded distal myocardium. After occlusion for 30 minutes, reperfusion occurred by releasing the ligature and removing the PE-10 tube. This allowed reperfusion of the formerly ischemic area.. 48.

(50) Figure. 2 Cardiac microcirculation in response to intravenous saline. Cardiac microcirculation does not change in response to intravenous in three genotype mice.. 49.

(51) Figure. 3 Cardiac microcirculation in response to intravenous U46619 (TP agonist, 2 mg/kg). In response to intravenous U46619, a decrease of cardiac microcirculation (30 sec) is found in B6 and TXAS-/-TP+/+ mice, but the cardiac microcirculation is not changed in TXAS-/-TP-/- mice. This decreased response is recovered within three minutes.. 50.

(52) Figure. 4 Cardiac microcirculation in response to intravenous ET-1 (2.5 µg/kg). Cardiac microcirculation is not altered in response to the low dose of ET1 among three genotype mice.. 51.

(53) Figure. 5 Cardiac microcirculation in response to intravenous ET-1 (25 µg/kg). In response to intravenous ET-1, the level of cardiac microcirculation is mildly decreased with 30 sec in B6 but that is not affected in TXAS-/-TP-/and TXAS-/-TP+/+ mice. A decrease in cardiac microcirculation quickly recovered within three minutes.. 52.

(54) Figure. 6 Cardiac microcirculation under intravenous ET-1 (250 µg/kg) in a minute. In response to intravenous ET-1, the level of cardiac microcirculation is mildly decreased with 30 sec in B6 but that is not affected in TXAS-/-TP-/and TXAS-/-TP+/+ mice. A decrease in cardiac microcirculation quickly recovered within three minutes.. 53.

(55) (a). (b). Figure. 7 (a) Response of perfusion unit of cardiac microcirculation to intravenous saline (red arrow) among three genotype mice. (b) The mean change of perfusion unit in response to saline among mice genotypes. In response to intravenous saline, there is no difference in perfusion unit among three genotype mice. 54.

(56) (a). (b). Figure. 8 (a) Perfusion unit of cardiac microcirculation under intravenous U46619 (TP agonist, 2mg/kg, red arrow) among three genotype mice. (b) The mean change of percentage of perfusion unit among mice genotypes. Mean ± SEM; N=4-5; *P < 0.05 vs. B6; aP < 0.05 vs. TXAS-/-TP+/+ mice. 55.

(57) (a). (b). Figure. 9 (a) Response of perfusion unit of cardiac microcirculation under intravenous ET-1 (2.5µg/kg, 25 µg/kg and 250 µg/kg, red, blue and green arrow) among three genotype mice. (b) The mean change of percentage of perfusion unit among mice genotypes. Mean ± SEM; N=3; * P < 0.05 vs. B6.. 56.

(58) B6. TXAS-/-TP+/+. TXAS-/-TP-/-. -1. 0. 1. 2. 3 min 0.1ms. Figure. 10 Response of ECG of three genotype mice under intravenous normal saline within three minutes. There is no difference in ECG when treatment with normal saline among three genotype mice.. 57.

(59) B6. TXAS-/-TP+/+. TXAS-/-TP-/-. -1. 0. 1. 2. 3 min 0.1ms. Figure. 11 Response of ECG of three genotype mice under intravenous U46619 (TP agonist, 2 mg/kg) within three minutes. Bradycardia or arrhythmia was found in B6 and TXAS-/-TP+/+ but not in TXAS-/-TP-/- mice when treatment with U46619. After U46619 challenge, TXAS-/-TP+/+ mice recovered normal ECG immediately within three minutes, however, B6 mice required more time to recover normal ECG.. 58.

(60) B6. TXAS-/-TP+/+. TXAS-/-TP-/-. 0. 2.5. 25. 250 µg/kg 0.1ms. Figure. 12 Response of ECG of three genotype mice under intravenous ET-1 (2.5, 25 and 250 µg/kg) among three genotype mice. There is no change in ECG among three genotype mice when treatment with low and middle dose of ET-1. Bradycardia or arrhythmia was found in B6 with high dose of ET-1.. 59.

(61) (a). (b). (c). Figure. 13 The R-R interval under intravenous (a) normal saline, (b) U46619 (2 mg/kg) and (c) ET-1 (2.5, 25 and 250 µg/kg) among three genotype mice. Mean ± SEM; N=3-5; *P < 0.05 compared to the B6 with saline control; #P < 0.05 compared to B6 with 250 µg/kg treatment.. 60.

(62) (a) (c). (b). (b). 61.

(63) Figure. 14 Effect of contraction and relaxation in mesenteric arteries. (a) Norepinephrine (b) Acetylcholine (c) U46619 (d) Endothelin-1. (Mean ± SEM.; N=6; *, B6+ASP and TXAS-/-TP-/- compared to B6, p<0.05; a, B6+ASP and TXAS-/-TP-/-compare to TXAS-/-TP+/+, p<0.05). 62.

(64) (a). Baseline. Ischemia 1 min. Ischemia 30 min. Reperfusion 1 min Reperfusion 120 min. (b). Reperfusion 1 min. Baseline. Reperfusion 120 min. Ischemia 1 min. Ischemia 30 min. Figure. 15 Response of cardiac microcirculation (a) and ECG (b) to myocardial ischemia/reperfusion injury in B6 mice.. 63.

(65) (a). Baseline. Ischemia 1 min. Ischemia 30 min. Reperfusion 1 min Reperfusion 120 min. (b). Reperfusion 1 min. Baseline. Ischemia 1 min. Reperfusion 120 min. Ischemia 30 min. Figure. 16 Response of cardiac microcirculation (a) and ECG (b) with myocardial ischemia/reperfusion injury in TXAS-/-TP+/+ mice.. 64.

(66) (a). Baseline. Ischemia 1 min. Ischemia 30 min. Reperfusion 1 min Reperfusion 120 min. (b). Reperfusion 1 min. Baseline. Ischemia 1 min. Reperfusion 120 min. Ischemia 30 min. Figure. 17 Response of cardiac microcirculation (a) and ECG (b) with myocardial ischemia/reperfusion injury in TXAS-/-TP-/- mice.. 65.

(67) (a). (b). Figure. 18 Histological feature of the heart with or without IR injury in three genotype mice. (a) The heart histological structure. Split formation was found among three genotype mice (red arrows, 400 x). (b) The mean data of percentage of split formation in these three groups of mice. Mean ± SEM; N=6; *P < 0.05 vs. Control; #P < 0.05 vs. B6 with IR injury.. 66.

(68) (a). (b). Figure. 19 Terminal deoxynucleotide transferase dUTP Nick End Labeling stain. (a) I/R increased myocardial cell apoptosis among three groups of mice. Apoptotic cells are expressed in three genotype mice with I/R injury (red arrow, 400 x) (b) The ratio of TUNEL stain after I/R is highest in B6, but is significantly reduced in TXAS-/-TP-/- and TXAS-/-TP+/+. (Mean ± SEM; N=6; **P < 0.01 vs. respective control; #P < 0.05 vs. B6 I/R.. 67.

(69) (a). (b). Figure. 20 Immunohistochemistry of Beclin-1. (a) I/R increased myocardial Beclin-1 autophagy expression (brown color) among three groups of mice. (b) The ratio of Beclin-1 stain after I/R is highest in B6, but is significantly reduced in TXAS-/-TP-/- and TXAS-/-TP+/+. (Mean ± SEM; N=6; *P < 0.05 vs. respective control; #P < 0.05 vs. B6 I/R.. 68.

(70) (a). (b). Figure. 21 Immunohistochemistry of IL-1β (a) I/R increased myocardial cell IL-1β expression among three groups of mice. Positive IL-1β stains are expressed in three genotype mice with I/R injury (b) The IL-1β stain after I/R is highest in B6, but is significantly reduced in TXAS-/-TP-/- and TXAS-/-TP+/+. (Mean ± SEM; N=6; *P < 0.05 vs. respective control; #P < 0.05 vs. B6 I/R.. 69.

(71) (a). (b). Figure. 22 Immunohistochemistry of 4-HNE (a) An increase of 4-HNE stain, a brown color indicated by red arrows, is increased in the myocardial cells of I/R B6 mice. 4-HNE expressed in three genotypes. (400 x) (b) The ratio of IL-1β stain across mice genotypes with I/R. (Mean ± SEM; N=6; *P < 0.05, compared to respective control; # P < 0.05 compared to I/R B6). 70.

(72) Figure. 23 Plasma Troponin-I concentration after I/R. Plasma Troponin-I levels with I/R injury in three knock out genotype mice. (Mean ± SEM; N=3; **, compared to respective control, P < 0.01; *** P < 0.001; # P < 0.05 compared to I/R B6). 71.

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