ATS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Ann Thorac Surg 2009;87:786-793. doi:10.1016/j.athoracsur.2008.12.021
© 2009 The Society of Thoracic Surgeons
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Personal Folders
Right arrow Download to citation manager
Right arrow Author home page(s):
Frank W. Sellke
Right arrow Permission Requests
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Osipov, R. M.
Right arrow Articles by Sellke, F. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Osipov, R. M.
Right arrow Articles by Sellke, F. W.
Related Collections
Right arrow Molecular biology
Right arrow Myocardial infarction

Original Articles: Adult Cardiac

Thrombin Fragment (TP508) Decreases Myocardial Infarction and Apoptosis After Ischemia Reperfusion Injury

Robert M. Osipov, MDa, Cesario Bianchi, MD, PhDa, Richard T. Clements, PhDa, Jun Feng, MD, PhDa, Yuhong Liu, MDa, Shu-Hua Xu, PhDa, Michael P. Robich, MDa, John Wagstaff, PhDb, Frank W. Sellke, MDa,*

a Department of General Surgery, Cardiothoracic Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
b OrthoLogic Corp, Tempe, Arizona

Accepted for publication December 5, 2008.

Abbreviations and Acronyms AAR = area at risk; Bcl 2 = B-cell lymphoma 2; BNIP 3 = Bcl2/adenovirus E1B 19 kDa-interacting protein; eNOS = endothelial nitric oxide synthase; HSP = heat shock protein; IR = ischemia-reperfusion; LAD = left anterior descending coronary artery; LV = left ventricle; PAR = protease-activated receptor; PARP = poly adenosine diphosphate-ribose polymerase; TUNEL = dUTP nick-end labeling

* Address correspondence to Dr Sellke, 110 Francis St, LMOB 2A, Boston, MA 02215 (Email: fsellke{at}caregroup.harvard.edu).


Dr Sellke discloses a financial relationship with Novo Nordisk.

 

   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Background: Myocardial ischemia-reperfusion injury may lead to cardiac dysfunction or death. This study investigates the potential efficacy of a novel thrombin fragment (TP508) on myocardial ischemia-reperfusion injury.

Methods: Fourteen male Yucatan pigs underwent 60 minutes of mid-left anterior descending coronary artery occlusion followed by 120 minutes of reperfusion. Pigs received either saline vehicle (control, n = 7) or thrombin fragment TP508 (n = 7) as a bolus (0.5 mg/kg) 50 minutes into the ischemic period, followed by continuous intravenous infusion (1.25 mg · kg–1 · h–1) during reperfusion. Myocardial function was monitored throughout the experiments. Monastryl blue/triphenyl tetrazolium chloride staining was utilized to measure the area at risk and infarcted tissue. Apoptosis was assessed by Western blotting and dUTP nick-end labeling (TUNEL) staining. Coronary microvascular reactivity to endothelium-dependent factors (adenosine diphosphate, substance P, A23187) and endothelium-independent factor (sodium nitroprusside) was examined.

Results: Global and regional left ventricular function was not significantly different between groups. Endothelium-dependent coronary microvascular relaxation was greater in the TP508 group and associated with higher endothelial nitric oxide synthase phosphorylation. Both infarct size and TUNEL staining was significantly decreased in the TP508 group compared with the control group (p < 0.05). Expression of the cell survival proteins B-cell lymphoma 2 (2.2-fold, p < 0.05) and heat shock protein-73 (1.6-fold, p < 0.05) was higher in the TP508 group. Expression of the cell-death–signaling proteins poly adenosine diphosphate-ribose polymerase (1.6-fold, p < 0.05), cleaved poly adenosine diphosphate-ribose polymerase (6.4-fold, p < 0.05), and B-cell lymphoma 2/adenovirus E1B 19 kDa-interacting protein 3 (3.8-fold, p < 0.05) was significantly higher in the TP508 group in the ischemic territory.

Conclusions: This study demonstrates that TP508 decreases infarct size, improves endothelial microvascular function, and induces cell-survival signaling in the setting of ischemia-reperfusion injury. Thus, TP508 may be a useful agent to attenuate myocardial reperfusion injury.

Despite the considerable progress made in pharmacologic and interventional management, acute myocardial infarction remains a leading cause of morbidity and mortality in the United States [1]. Prompt reperfusion, either with thrombolysis, primary percutaneous transluminal coronary angioplasty, or coronary artery bypass grafting, is essential to salvage ischemic myocardium. However, these interventions may induce iatrogenic damage, commonly referred to as ischemia-reperfusion (IR) injury [2].

Previous studies have shown that IR injury causes activation of thrombin with subsequent myocardial damage [3]. Deleterious effects of thrombin are mediated through activation of a family of protease-activated receptors (PAR) [4], which lead to the expression of cytokines, chemokines, and adhesion molecules in the myocardium [5–7]. Antithrombin therapies or inhibition of PAR receptors have been shown to ameliorate myocardial IR injury [8, 9].

The thrombin fragment TP508 (also known as rusalatide acetate or Chrysalin) is a 23-amino acid peptide that represents a portion of the highly preserved, noncatalytic site of the receptor binding domain in the native thrombin molecule. Recently, several animal studies have suggested that TP508 may offer protection of the myocardium during IR injury [10–12]. Currently, the exact mechanisms of TP508-induced responses are unclear, but TP508 interacts with high-affinity binding sites found on fibroblasts, neutrophils, monocytes, endothelial, and epithelial cells [13]. Studies conducted in human umbilical vein endothelial cell cultures have shown that TP508 is able to antagonize thrombin-induced deleterious cellular effects through specific {alpha}vβ3 integrin binding in an arginine-glycine-aspartic acid sequence–dependent manner. In addition, TP508 alone can induce protective responses during surgically induced ischemia after closure of dermal excisions through upregulation of inflammatory mediators, early growth factors, and improved angiogenesis [14, 15]. In light of these studies, we hypothesized that TP508, either through induction of protective responses or antagonistic actions on native thrombin signaling, may offer potential benefit in ameliorating myocardial IR injury. Therefore, we evaluated TP508 in a clinically relevant large animal model of acute IR injury.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Animals
Five-month-old intact Yucatan male mini-swine (average 23 kg) were housed individually and provided with laboratory chow and water ad libitum. All experiments were approved by the Beth Israel Deaconess Medical Center Animal Care and Use Committee and the Harvard Medical Area Standing Committee on Animals and conformed to the US National Institutes of Health guidelines regulating the care and use of laboratory animals (NIH publication 5377-3, 1996).

Surgical Protocol
Pigs were sedated with Telazol (Fort Dodge Animal Health, Fort Dodge, IA) before induction of anesthesia (1.5 mg/kg, intramuscularly) followed by endotracheal intubation and ventilation with a volume-cycled ventilator (North American Dragger, Telford, PA). General endotracheal anesthesia was established and maintained with 2.0% isoflurane (Ultane; Abbott Laboratories, Abbott Park, IL). The right common femoral artery and vein were canulated with a 7F Cordis sheath and 7F triple-lumen catheter, respectively (Cordis Corporation, Miami, FL). The right common femoral artery was used for arterial blood sampling and continuous monitoring of intra-arterial blood pressure (Millar Instruments, Houston, TX). Arterial blood gas analysis, hematocrit, and core temperature were assessed every 30 minutes. Before left anterior descending coronary artery (LAD) occlusion, each animal received a 1-L bolus of Lactated Ringer's solution followed by infusion at a rate of 15 mL · kg–1 · h–1. A phenylephrine drip (0.25 µg · kg–1 · min–1) to prevent hypotension induced by isofluorane, heparin (80 units/kg bolus), and lidocaine (1.5 mg/kg) to control ventricular dysrhythmia, were administered.

A median sternotomy was performed, and a pericardial cradle was created. A catheter-tipped manometer (Millar Instruments, Houston, TX) was introduced through the apex of the left ventricle (LV) to record LV pressure. The LAD was occluded 3 mm distal to the origin of the second diagonal branch by a Rommel tourniquet. After 60 minutes of ischemia, the tourniquet was released, and the myocardium was reperfused for 120 minutes. Reperfusion was monitored utilizing a transonics Doppler probe on the LAD. At the end of the reperfusion period, the LAD was religated and monastryl blue pigment diluted in phosphate-buffered saline (1:150) (Engelhard Corp, Louisville, KY) was injected into the aortic root to demarcate the area at risk (AAR). The heart was rapidly excised and sliced into 1-cm-thick slices perpendicular to the axis of the LAD up to the area of LAD ligation. Tissue from the second slice was isolated for use in molecular and coronary microvascular reactivity studies. The remaining tissue was incubated in a 1% triphenyl tetrazolium chloride (TTC; Sigma Chemical, St Louis, MO) solution in phosphate-buffered saline for 30 minutes, and infarct size was assessed as previously described [16]. Ventricular dysrhythmia (ventricular fibrillation or pulseless ventricular tachycardia) events were recorded and treated with immediate electrical cardioversion with 20 to 50 J.

Experimental Design
Pigs received either vehicle solution (control, n = 7) or TP508 peptide (TP508, n = 7), administered intravenously as a bolus (0.5 mg/kg, over 2 minutes) 50 minutes into ischemia, followed by continuous infusion (1.25 mg · kg–1 · h–1) during reperfusion using an infusion pump (Harvard Apparatus, Hollistone, MA).

Measurement of Global and Regional Myocardial Function
Indices of global and regional myocardial function were monitored during the entire experiment. Monitored measurements were mean arterial pressure, developed LV pressure, and positive (+dP/dt) and negative (–dP/dt) first derivatives of LV pressure. Segmental shortening in the AAR was measured through ultrasonic crystals (Sonometrics, London, ON, Canada). Global and regional functional measurements recorded for 10 sequential beats at baseline and then every half hour (occlusion 1 = 30 minutes, occlusion 2 = 60 minutes; reperfusion 1 = 30, reperfusion 2 = 60, reperfusion 3 = 90, and reperfusion 4 = 120 minutes) using a Sonometrics system as previously described [17].

Quantification of Myocardial Infarct Size
One-centimeter slices of whole heart were incubated in a 1% TTC solution as previously described [17]. Briefly, necrotic (pale), the AAR (bright red), and nonischemic portions of the heart specimens (purple) were photographed and measured. Left ventricular infarct size was calculated as a percentage of AAR necrosis in each individual slice with planimetry (Image J 1.4; National Institutes of Health, Bethesda, MD) using the following equation: [(LV infarct surface area/LV AAR surface area) x 100]. The AAR was calculated using the following equation: [(LV infarct area + noninfarct AAR)/LV total surface x 100].

Coronary Microvascular Reactivity Studies
Coronary microvascular reactivity was examined in samples from the ischemic territory as previously described [17]. Briefly, coronary arterioles (80 µm to 130 µm) were dissected with a x40 microscope. Microvessels were mounted on dual glass micropipettes and examined in a pressurized isolated microvessel chamber. Adenosine diphosphate (ADP [1 nM to 100 uM]), substance P (0.1 pM to 10 nM), A23187 (1 nM to 10 uM), and sodium nitroprusside (1 nM to 100 µM) were applied extraluminally after precontraction by 20% to 50% of the baseline diameter with the thromboxane A2 analog U46619 (0.1 µM to 1 µM).

Western Blotting
Myocardial samples were homogenized in RIPA buffer (Boston Bioproducts, Worcester, MA), and protein concentration was measured using a BCA assay (Pierce, Rockford, IL). Then 40 µg to 60 µg lysate were subjected to SDS-Page and immunoblotting as previously described [17]. Primary antibodies for immunoblots were used according to the manufacturer's recommendation. Levels of B-cell lymphoma 2 (Bcl2), caspase-3, cleaved caspase-3, poly adenosine diphosphate-ribose polymerase (PARP), cleaved PARP, Bcl2/adenovirus E1B 19 kDa-interacting protein (BNIP3), endothelial nitric oxide synthase (eNOS) and phospho-eNOS (Ser1177; Cell Signaling Technology, Beverly, MA), and heat shock protein (HSP)-73 (Assay Designs, Ann Arbor, MI) were assessed. Ponceau staining was used to confirm equal protein loading and to normalize protein expression. Data are presented as mean ± SEM in arbitrary density units.

TUNEL Staining
Apoptotic cells were identified by dUTP nick-end labeling (TUNEL) using an apoptosis detection kit (Chemicon, Temecula, CA). At least 1 cm2 tissue from the AAR was analyzed from each animal (4 per group). The number of TUNEL-positive cardiomyocytes is expressed as mean/cm2.

Statistical Analysis
Functional and microvascular reactivity data were analyzed using two-way, repeated measures analysis of variance. Infarct size and densitometry were analyzed using Student's t test. The {chi}2 test was used to determine any significant difference in the incidence of fibrillation. Data are reported as mean ± SEM. All p values less than 0.05 were considered significant for all statistical tests (Systat, San Jose, CA).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Arterial Blood Gas, Hematocrit, and Core Temperature
No significant differences were observed between groups with regard to arterial pH, pCO2, pO2, hematocrit, or core temperature at baseline or at the end of reperfusion.

Global Left Ventricular Function
The mean arterial blood pressure, developed LV pressure, positive (+dP/dt) and negative (–dP/dt) first derivatives of LV pressure were similar between groups at baseline and at the end of reperfusion (Fig 1A to D).


Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		Fig 1. (A) Mean arterial pressure (p = 0.5), (B) developed left ventricular pressure (p = 0.4), (C) positive first derivative of left ventricular pressure (+dP/dt [p = 0.6]), and (D) negative first derivative of left ventricular pressure (–dP/dt [p = 0.8]) in control group (diamonds) versus TP508 group (squares).

 
Regional Left Ventricular Function
Regional myocardial function in the AAR was similar between groups in both longitudinal and horizontal axes at all time points examined. There were significant decreases in segmental shortening relative to baseline function during all time points of ischemia and reperfusion (Fig 2A and B).


Figure 2
View larger version (29K):
[in this window]
[in a new window]

  		Fig 2. Regional left ventricular function: (A) Longitudinal axis (p = 0.6) and (B) horizontal axis segmental shortening (p = 0.9) in control group (diamonds) versus TP508 group (squares).

 
Incidence of VF/VT
There was no significant difference between groups in incidence of ventricular fibrillation or pulseless ventricular tachycardia (control 4 of 7 versus TP508 2 of 7, p = 0.3). All dysrrythmias were successfully terminated with electrical cardioversion.

Myocardial Infarct Size
Although AAR was similar between groups, the area of infarction was significantly decreased in the TP508 group versus control (Fig 3A and B). In the control group, 5 of 7 specimens had transmural necrosis involving the LV wall and septum compared with 3 of 7 in the TP508 group (p = 0.3). In all cases, the endocardium was preserved. The infarct was confirmed by hematoxylin and eosin staining.


Figure 3
View larger version (95K):
[in this window]
[in a new window]

  		Fig 3. (A) Percent of left ventricle wall area at risk (AAR) and (B) percent of infarction of the AAR in control group (open bars) versus TP508 group (shaded bars). The pictures represent the third slice from (C) a control animal and (D) a TP508 animal, after triphenyl tetrazolium chloride staining. Three zones can be differentiated: nonischemic area (purple), area at risk (bright red), and necrotic area (pale). White arrows indicate infarct area, with transmural infarct involving anterior wall of left ventricle and septum in the control animal; the red arrow indicates nonnecrotic endocardium. *p < 0.05.

 
Left Anterior Descending Coronary Artery Blood Flow
Although the LAD blood flow (Fig 4) was not significantly different between groups, release of occlusion caused a spike of LAD flow in the first minute of reperfusion that was greater in the TP508 group (2.7-fold versus 2.3-fold over baseline, p < 0.05).


Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		Fig 4. Left anterior descending coronary artery blood flow (p = 0.7) in control (diamonds) and TP508 (squares) animals. The spike during first minutes into reperfusion was due to the phenomenon known as ischemic hyperemia. (Pre = before occlusion; R = reperfusion.)

 
Coronary Microvascular Reactivity in the Ischemic Territory
Receptor-mediated endothelium-dependent responses to ADP and substance P, and nonreceptor-mediated endothelium-dependent responses to A23187 were significantly greater in the TP508 group compared with the control group (Fig 5A, B, and C). There was no significant difference between groups in response to the endothelium-independent vasodilator sodium nitroprusside (Fig 5D). Although the expression of eNOS was similar in both groups, the expression of phospho-eNOS (Ser1177) was significantly higher in the TP508-treated group (Fig 5E).


Figure 5
View larger version (32K):
[in this window]
[in a new window]

  		Fig 5. Microvascular responses of control (diamonds) and TP508 (squares) animals to endothelium-dependent agents: (A) adenosine diphosphate (ADP), (B) substance P, and (C) calcium ionophore A23187. (D) Microvascular response to endothelium-independent agent sodium nitroprusside (SNP) in control versus TP505 groups (p = 0.2). Data presented as percent of relaxation in control group versus TP508 group. (E) Western blot for phospho-endothelial nitric oxide synthase (p-eNOS [Ser1177]) normalized to total eNOS in control group (open bar [n = 6]) versus TP508 group (shaded bar [n = 6]). Insets are representative Western blots. *p < 0.05.

 
Profile of Cell Survival Signals
The expression of antiapoptotic Bcl-2 (Fig 6A) and HSP-73 (Fig 6B) were significantly higher in the TP508 group versus control group.


Figure 6
View larger version (31K):
[in this window]
[in a new window]

  		Fig 6. Western blot for (A) B-cell lymphoma 2 (Bcl-2) and (B) and heat shock protein (HSP)-73 in control group (open bars [n = 7]) versus TP508 group (shaded bars [n = 7]). Insets are representative Western blots. *p < 0.05.

 
Profile of Cell Death Signals
The expression of proapoptotic proteins BNIP-3 (Fig 7A), PARP and its inactive form, cleaved PARP (Fig 8A and B) were greater in the TP508 group compared with control. Ischemia was associated with greater expression of cleaved caspase-3 in both groups (Fig 7B).


Figure 7
View larger version (37K):
[in this window]
[in a new window]

  		Fig 7. Western blot for (A) B-cell lymphoma 2/adenovirus E1B 19 kDa-interacting protein (BNIP-3) and (B) and cleaved caspase-3 in control group (open bars [n = 7]) versus TP508 group (shaded bars [n = 7]). Cleaved caspase-3 expression was normalized to total caspase-3. Insets are representative Western blots. *p < 0.05.

 

Figure 8
View larger version (32K):
[in this window]
[in a new window]

  		Fig 8. Western blot for (A) poly adenosine diphosphate-ribose polymerase (PARP) and (B) cleaved PARP in control group (open bars [n = 7]) versus TP508 group (shaded bars [n = 7]). Insets are representative Western blots. *p < 0.05.

 
TUNEL Staining for Apoptosis
Apoptotic cell counts were 1.8-fold (p < 0.05) higher in the control group (12 ± 1.5 TUNEL-positive cells/cm2) versus the TP508 group (6.8 ± 1.2 TUNEL-positive cells/ cm2).


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
The key findings of this study are that TP508 provides significant myocardial protection in the setting of IR injury. The thrombin peptide TP508 administered before reperfusion reduces infarct size, improves microvascular function, and induces the expression of cell survival proteins.

Although TP508 significantly decreases myocardial infarction, we did not see any differences in global and regional LV function between groups. One potential reason for this apparent discrepancy is that thrombin can depress cardiomyocyte contractility and interfere with β-adrenergic responsiveness [18]. It is possible that this function of thrombin may be mimicked by administration of TP508. Thus, it may be that the acute initial protective effects of TP508 fail to be translated owing to transient negative inotropic effects. Another possibility is that the nonnecrotic myocardium in the area at risk exhibits ischemia-induced myocardial stunning [19, 20]. Thus, the salvaged yet stunned myocardium in the TP508 group may exhibit similar functional attributes as the necrotic tissue in the control group. In support, the AAR was similar between groups, which would result in similar amounts of stunned or necrotic tissue and may account for the lack of difference in cardiac function. Future studies examining long-term functional improvements (i.e., more than 2 hours) will be required to determine if the TP508-dependent decrease in necrotic tissue results in improved cardiac function after myocardial infarction.

Treatment with TP508 also resulted in a significant improvement in endothelial-dependent coronary microvascular relaxation. This improvement in the TP508 group is likely due to a significant induction of eNOS phosphorylation resulting in a increase bioavailable nitric oxide (NO) or due to less ischemic injury. Our findings are consistent with a recent report demonstrating that TP508 improves endothelial dysfunction, and induces phosphorylation of eNOS and NO production in a porcine model of chronic myocardial ischemia [21]. Furthermore, thrombin has been shown to induce eNOS phosphorylation through Ca2+mobilization and protein kinase C activation [22], enhancing the release of vasodilator molecules with protective properties [23], and TP508 may potentially work through the same pathway. The TP508-dependent increase in vasodilation may enhance subsequent reperfusion and limit the extent of myocardial necrosis after IR injury.

The delicate balance between cell death and cell survival proteins plays a crucial role in cell survival after myocardial IR injury. Surprisingly, TP508 treatment induced activation of both cell death and cell survival signaling mechanisms: TP508 increased caspase cleavage, PARP expression and cleavage, and expression of BNIP-3. Capase cleavage is implicated in both necrotic and apoptotic cell death pathways; PARP is a nuclear enzyme activated by DNA damage and overactivation of PARP results in the depletion of nicotinamide adenine dinucleotide and subsequent adenosine triphosphate formation, leading to cell death [24]. Activated BNIP-3 is implicated in caspase-independent necrosis and cell death [25] through its ability to regulate mitochondrial permeability transition pore sites [26].

In contrast to cell death signaling cascades, TP508 increased the expression of the cell survival proteins HSP-73 and Bcl-2. The HSP-70 stress responsive protein family is capable of binding and sequestering activated caspases and apoptosis-inducing factor [27–30], leading to improved cell survival. In addition, HSP-70 family proteins are known to play an important role in cell survival by stabilizing protein structure during stress. Moreover, Bcl-2 is able to block opening of mitochondrial permeability transition pore sites [31], release of mitochondrial cytochrome c, elaboration of apoptosis-inducing factor, and activation of executioner caspases-3, -6, and -7 [32, 33]; and Bcl-2 has also been shown to act as a free radical scavenger [34]. Therefore, induction of these proteins by TP508 may be sufficient to counteract the expression of the above-mentioned cell death signals.

One possibility for the induction of prosurvival signals and improved myocardial salvage in the face of increased apoptotic signals is that TP508 may result in a form of preconditioning that initiates prosurvival pathways. Recent studies have shown that during brief chemical preconditioning, moderate increases in the expression of proapoptotic signals (i.e., caspase cleavage) cause upregulation of expression of prosurvival proteins such as HSP-73 [35, 36]. Interestingly, TP508 treatment resulted in increased caspase cleavage in the nonischemic myocardium. This finding suggests that activation of caspase-3 in the TP508-treated animals may indeed lead to a similar mechanism of enhanced Bcl-2 and HSP-73 expression in cardiomyocytes [35–37] (Fig 9).


Figure 9
View larger version (22K):
[in this window]
[in a new window]

  		Fig 9. Proposed mechanism of TP508-induced myocardial protection in response to ischemia-reperfusion (IR) injury. (Bcl-2 = B-cell lymphoma 2; BNIP-3 = Bcl-2/adenovirus E1B 19 kDa-interacting protein; HSP = heat shock protein; PARP = poly adenosine diphosphate-ribose polymerase.)

 
In conclusion, the findings of this study demonstrate that parenteral administration of the TP508 provides significant myocardial and coronary microvascular protection in the setting of acute IR injury. It appears that these beneficial effects may be related to the higher expression of cell survival signals. Thus, TP508 may have a therapeutic role in limiting myocardial injury after acute myocardial infarction and cardiac surgery.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
We thank the staff of the Animal Research Facility at the Beth Israel Deaconess Medical Center for their efforts. Funding for this project was provided by grants NHLBI-RO1, RO1HL46716, RO1HL69024, RO1HL85647, T32-HL076130, and by grants from OrthoLogic Corp, Tempe, Arizona (to F.W.S.), and the Irving Bard Memorial Fellowship (to R.M.O.).


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Murray CJ, Lopez AD, Wibulpolprasert S. Monitoring global health: time for new solutions Br Med J 2004;329:1096-1100.[Free Full Text]
  2. Gottlieb S, Leor J, Shotan A, et al. Comparison of effectiveness of angiotensin-converting enzyme inhibitors after acute myocardial infarction in diabetic versus nondiabetic patients Am J Cardiol 2003;92:1020-1025.[Medline]
  3. Raivio P, Kuitunen A, Suojaranta-Ylinen R, Lassila R, Petaja J. Thrombin generation during reperfusion after coronary artery bypass surgery associates with postoperative myocardial damage J Thromb Haemost 2006;4:1523-1529.[Medline]
  4. Dery O, Corvera CU, Steinhoff M, Bunnett NW. Proteinase-activated receptors: novel mechanisms of signaling by serine proteases Am J Physiol 1998;274:C1429-C1452.[Medline]
  5. Chong AJ, Pohlman TH, Hampton CR, Shimamoto A, Mackman N, Verrier ED. Tissue factor and thrombin mediate myocardial ischemia-reperfusion injury Ann Thorac Surg 2003;75(Suppl):649-655.
  6. Erlich JH, Boyle EM, Labriola J, et al. Inhibition of the tissue factor-thrombin pathway limits infarct size after myocardial ischemia-reperfusion injury by reducing inflammation Am J Pathol 2000;157:1849-1862.[Medline]
  7. Kranzhofer R, Clinton SK, Ishii K, Coughlin SR, Fenton JW, Libby P. Thrombin potently stimulates cytokine production in human vascular smooth muscle cells but not in mononuclear phagocytes Circ Res 1996;79:286-294.[Abstract/Free Full Text]
  8. Jormalainen M, Vento AE, Lukkarinen H, et al. Inhibition of thrombin during reperfusion improves immediate postischemic myocardial function and modulates apoptosis in a porcine model of cardiopulmonary bypass J Cardiothorac Vasc Anesth 2007;21:224-231.[Medline]
  9. Gudmundsdottir IJ, Lang NN, Boon NA, et al. Role of the endothelium in the vascular effects of the thrombin receptor (protease-activated receptor type 1) in humans J Am Coll Cardiol 2008;51:1749-1756.[Abstract/Free Full Text]
  10. Ryaby JT, Sheller MR, Levine BP, Bramlet DG, Ladd AL, Carney DH. Thrombin peptide TP508 stimulates cellular events leading to angiogenesis, revascularization, and repair of dermal and musculoskeletal tissues J Bone Joint Surg Am 2006;88(Suppl 3):132-139.[Medline]
  11. Norfleet AM, Huang Y, Sower LE, Redin WR, Fritz RR, Carney DH. Thrombin peptide TP508 accelerates closure of dermal excisions in animal tissue with surgically induced ischemia Wound Repair Regen 2000;8:517-529.[Medline]
  12. Stiernberg J, Norfleet AM, Redin WR, Warner WS, Fritz RR, Carney DH. Acceleration of full-thickness wound healing in normal rats by the synthetic thrombin peptide, TP508 Wound Repair Regen 2000;8:204-215.[Medline]
  13. Glenn KC, Frost GH, Bergmann JS, Carney DH. Synthetic peptides bind to high-affinity thrombin receptors and modulate thrombin mitogenesis Pept Res 1988;1:65-73.[Medline]
  14. Wang H, Li X, Tomin E, et al. Thrombin peptide (TP508) promotes fracture repair by up-regulating inflammatory mediators, early growth factors, and increasing angiogenesis J Orthop Res 2005;23:671-679.[Medline]
  15. Norfleet AM, Bergmann JS, Carney DH. Thrombin peptide, TP508, stimulates angiogenic responses in animal models of dermal wound healing, in chick chorioallantoic membranes, and in cultured human aortic and microvascular endothelial cells Gen Pharmacol 2000;35:249-254.[Medline]
  16. Toyoda Y, Di Gregorio V, Parker RA, Levitsky S, McCully JD. Anti-stunning and anti-infarct effects of adenosine-enhanced ischemic preconditioning Circulation 2000;102(Suppl 3):326-331.[Abstract/Free Full Text]
  17. Sodha NR, Clements RT, Feng J, et al. The effects of therapeutic sulfide on myocardial apoptosis in response to ischemia-reperfusion injury Eur J Cardiothorac Surg 2008;33:906-913.[Abstract/Free Full Text]
  18. Hird RB, Crawford FA, Mukherjee R, Spinale FG. Direct effects of thrombin on myocyte contractile function Ann Thorac Surg 1995;59:288-293.[Abstract/Free Full Text]
  19. Kloner RA, Arimie RB, Kay GL, et al. Evidence for stunned myocardium in humans: a 2001 update Coron Artery Dis 2001;12:349-356.[Medline]
  20. Bolli R, Becker L, Gross G, Mentzer R, Balshaw D, Lathrop DA. Myocardial protection at a crossroads: the need for translation into clinical therapy Circ Res 2004;95:125-134.[Abstract/Free Full Text]
  21. Fossum TW, Olszewska-Pazdrak B, Mertens MM, et al. TP508 (Chrysalin) reverses endothelial dysfunction and increases perfusion and myocardial function in hearts with chronic ischemia J Cardiovasc Pharmacol Ther 2008;13:214-225.[Abstract/Free Full Text]
  22. Motley ED, Eguchi K, Patterson MM, Palmer PD, Suzuki H, Eguchi S. Mechanism of endothelial nitric oxide synthase phosphorylation and activation by thrombin Hypertension 2007;49:577-583.[Abstract/Free Full Text]
  23. Syeda F, Grosjean J, Houliston RA, et al. Cyclooxygenase-2 induction and prostacyclin release by protease-activated receptors in endothelial cells require cooperation between mitogen-activated protein kinase and NF-kappaB pathways J Biol Chem 2006;281:11792-11804.[Abstract/Free Full Text]
  24. Boulares AH, Yakovlev AG, Ivanova V, et al. Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells. J Biol Chem 1999;274:22932-22940.[Abstract/Free Full Text]
  25. Regula KM, Ens K, Kirshenbaum LA. Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes Circ Res 2002;91:226-231.[Abstract/Free Full Text]
  26. Crompton M. Mitochondrial intermembrane junctional complexes and their role in cell death J Physiol 2000;529:11-21.[Abstract/Free Full Text]
  27. Lepore DA, Knight KR, Anderson RL, Morrison WA. Role of priming stresses and Hsp70 in protection from ischemia-reperfusion injury in cardiac and skeletal muscle Cell Stress Chaperones 2001;6:93-96.[Medline]
  28. Ravati A, Ahlemeyer B, Becker A, Krieglstein J. Preconditioning-induced neuroprotection is mediated by reactive oxygen species Brain Res 2000;866:23-32.[Medline]
  29. Beere HM, Wolf BB, Cain K, et al. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome Nat Cell Biol 2000;2:469-475.[Medline]
  30. Ohtsuka K, Hata M. Molecular chaperone function of mammalian Hsp70 and Hsp40–a review Int J Hyperthermia 2000;16:231-245.[Medline]
  31. Kroemer G, Dallaporta B, Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis Annu Rev Physiol 1998;60:619-642.[Medline]
  32. Stegh AH, Kesari S, Mahoney JE, et al. Bcl2L12-mediated inhibition of effector caspase-3 and caspase-7 through distinct mechanisms in glioblastoma Proc Natl Acad Sci USA 2008;105:10703-10708.[Abstract/Free Full Text]
  33. Okamura T, Miura T, Takemura G, et al. Effect of caspase inhibitors on myocardial infarct size and myocyte DNA fragmentation in the ischemia-reperfused rat heart Cardiovasc Res 2000;45:642-650.[Abstract/Free Full Text]
  34. Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis Cell 1993;75:241-251.[Medline]
  35. Garnier P, Ying W, Swanson RA. Ischemic preconditioning by caspase cleavage of poly(ADP-ribose) polymerase-1 J Neurosci 2003;23:7967-7973.[Abstract/Free Full Text]
  36. Kaushal GP, Basnakian AG, Shah SV. Apoptotic pathways in ischemic acute renal failure Kidney Int 2004;66:500-506.[Medline]
  37. Adrain C, Creagh EM, Martin SJ. Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2 EMBO J 2001;20:6627-6636.[Medline]



This article has been cited by other articles:


Home page
 Ann. Thorac. Surg.Home page
C. Zhang, J. Hou, S. Zheng, Z. Zheng, and S. Hu
Vascularized Atrial Tissue Patch Cardiomyoplasty With Omentopexy Improves Cardiac Performance After Myocardial Infarction
Ann. Thorac. Surg., October 1, 2011; 92(4): 1435 - 1442.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
L. M. Chu, R. M. Osipov, M. P. Robich, J. Feng, M. R. Sheller, and F. W. Sellke
Effect of Thrombin Fragment (TP508) on Myocardial Ischemia Reperfusion Injury in a Model of Type 1 Diabetes Mellitus
Circulation, September 14, 2010; 122(11_suppl_1): S162 - S169.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
S. Oyamada, R. Osipov, C. Bianchi, M. P. Robich, J. Feng, Y. Liu, T. A. Burgess, T. M. Bell, M. R. Sheller, and F. W. Sellke
Effect of Dimerized Thrombin Fragment TP508 on Acute Myocardial Ischemia Reperfusion Injury in Hypercholesterolemic Swine
J. Pharmacol. Exp. Ther., August 1, 2010; 334(2): 449 - 459.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
R. M. Osipov, M. P. Robich, J. Feng, R. T. Clements, Y. Liu, H. P. Glazer, J. Wagstaff, C. Bianchi, and F. W. Sellke
Effect of thrombin fragment (TP508) on myocardial ischemia-reperfusion injury in hypercholesterolemic pigs
J Appl Physiol, June 1, 2009; 106(6): 1993 - 2001.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Personal Folders
Right arrow Download to citation manager
Right arrow Author home page(s):
Frank W. Sellke
Right arrow Permission Requests
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Osipov, R. M.
Right arrow Articles by Sellke, F. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Osipov, R. M.
Right arrow Articles by Sellke, F. W.
Related Collections
Right arrow Molecular biology
Right arrow Myocardial infarction

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
ANN THORAC SURG ASIAN CARDIOVASC THORAC ANN EUR J CARDIOTHORAC SURG
J THORAC CARDIOVASC SURG ICVTS ALL CTSNet JOURNALS