VII. DISCUSSION
1. PREVIOUS STUDIES OF TXAS-TXA 2 -TP SIGNALING AND ET-1 IN
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.
32
Our experiments showed the change of heart microcirculation through TXAS-TXA2-TP signaling first time.
2. Impact of ECG and cardiac microcirculation through TXAS-TXA
2-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
33
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 TXAS-TXA
2-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
34
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+ASPand 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+ASPand 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-TXA
2-TP signaling attenuate injury evoked
by myocardial ischemia reperfusion through apoptosis, oxidative
35
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 presentedwhen 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 TXA
2with 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-TXA
2-TP signaling could reduce I/R injury in mouse heart. We cannot find any difference on heartmicrocirculation in this model among three genotype mice.
36
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-TP-
37
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). IL-1β 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 TXA2 -TP- signaling. Main impairment of reperfusion result from oxidative stress had been discussed a long time. Various studies
38
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.
39
VIII. Conclusion
In summary, this study can divide into two parts. (1) In order to elaborate the relationship between TXA2-TP- signaling and ET-1 on heart microcirculation and function. Inhibition of TXA2 -TP-signaling 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.
40
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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 1-mm section of PE-10 tubing placed on top of the LAD, 2 to 3 1-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.
49
Figure. 2 Cardiac microcirculation in response to intravenous saline.
Cardiac microcirculation does not change in response to intravenous in three genotype mice.
50
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.
51
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 ET-1 among three genotype mice.
52
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.
53
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.
54
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.(a)
(b)
55
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.
(a)
(b)
56
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.
(a)
(b)
57
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.0 -1
1
2
3 min
0.1ms
TXAS-/-TP+/+
B6 TXAS-/-TP
-/-58
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.0 -1
1
2
3 min
TXAS-/-TP+/+
B6 TXAS-/-TP
-/-0.1ms
59
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-1.2.5 0
25
250 µg/kg
TXAS-/-TP+/+
B6 TXAS-/-TP
-/-0.1ms
60
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.(a)
(b)
(c)
61
(b) (a) (c)
(b)
62
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)
63
Figure. 15 Response of cardiac microcirculation (a) and ECG (b) to myocardial ischemia/reperfusion injury in B6 mice.
Baseline Ischemia 1 min Ischemia 30 min
Reperfusion 1 min Reperfusion 120 min (a)
Reperfusion 120 min Ischemia 1 min
Ischemia 30 min
Baseline Reperfusion 1 min
(b)
64
Figure. 16 Response of cardiac microcirculation (a) and ECG (b) with myocardial ischemia/reperfusion injury in TXAS
-/-TP
+/+mice.
Baseline Ischemia 1 min Ischemia 30 min
Reperfusion 1 min Reperfusion 120 min (a)
(b)
Reperfusion 120 min Baseline
Ischemia 1 min
Ischemia 30 min
Reperfusion 1 min
65
Figure. 17 Response of cardiac microcirculation (a) and ECG (b) with myocardial ischemia/reperfusion injury in TXAS
-/-TP
-/-mice.
Baseline Ischemia 1 min Ischemia 30 min
Reperfusion 1 min Reperfusion 120 min (a)
(b) Baseline
Ischemia 1 min
Ischemia 30 min
Reperfusion 120 min
Reperfusion 1 min
66
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.(a)
(b)
67
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.(a)
(b)
68
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.
(a)
(b)
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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.
(a)
(b)
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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)
(a)
(b)
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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)