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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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Articles by Benedict R. Lucchesi in JoVE

 JoVE Clinical and Translational Medicine

Electrolytic Inferior Vena Cava Model (EIM) of Venous Thrombosis


JoVE 2737 7/12/2011

1, 1, 1, 2, 1, 1

1Conrad Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan, 2Department of Pharmacology, University of Michigan

The electrolytic induction of endothelial activation to the internal surface of the Inferior Vena Cava results in venous type thrombus formation due to endothelial activation and partial blood stasis, two components of Virchow's triad.

Other articles by Benedict R. Lucchesi on PubMed

Intimatan Prevents Arterial and Venous Thrombosis in a Canine Model of Deep Vessel Wall Injury

The Journal of Pharmacology and Experimental Therapeutics. Jun, 2002  |  Pubmed ID: 12023550

Resistance of fibrin-bound thrombin to inactivation by the heparin/antithrombin III complex is considered a limitation in the use of heparin as an antithrombotic agent. Intimatan (dermatan 4,6-di-O-sulfate) is a heparin cofactor II agonist that inhibits both free and bound forms of thrombin. The present study examines the hypothesis that Intimatan prevents thrombotic occlusion in response to vascular wall injury in a canine model of carotid artery/jugular vein thrombosis. The left carotid artery and right jugular vein served as vehicle-treated control vessels, whereas the right carotid artery and left jugular vein were subjected to electrolytic injury after administration of Intimatan (9 mg/kg bolus + 300 microg/kg/min infusion, i.v.) or dalteparin (Fragmin) (400 IU/kg, s.c.). Intimatan significantly increased time to carotid artery (226.0 +/- 14.0 min) and jugular vein (240.0 +/- 0.0 min) thrombosis, compared with control vessels (carotid artery, 87.1 +/- 7.9 min; jugular vein, 60.6 +/- 7.4 min). Vessel patency was maintained in eight of eight jugular veins and seven of eight carotid arteries during treatment with Intimatan. Dalteparin significantly increased time to carotid artery thrombosis (122.1 +/- 17.5 min) compared with control (64.3 +/- 8.2 min), but did not change the time to thrombosis in the jugular vein. Only one carotid artery remained patent at the end of the dalteparin protocol. The two drugs produced minimal increases in bleeding times, and Intimatan increased the activated partial thromboplastin time above that observed with dalteparin. The results demonstrate that Intimatan is effective in preventing occlusive arterial and venous thrombosis in an experimental model of deep vascular wall injury.

Prevention of Experimental Carotid and Coronary Artery Thrombosis by the Glycoprotein IIb/IIIa Receptor Antagonist CRL42796

British Journal of Pharmacology. Jul, 2002  |  Pubmed ID: 12110617

1. The antithrombotic effect of the glycoprotein IIb/IIIa receptor antagonist, CRL42796, was examined in canine models of carotid and coronary artery thrombosis. 2. In the carotid artery thrombosis model, occlusion occurred in all control vessels (time to thrombosis 47.6+/-8.9 min). After treatment with low dose CRL42796 (15 microg kg(-1) loading dose +0.31 microg kg(-1) min(-1) i.v.), two of five vessels occluded. Time to thrombosis increased significantly to 155.2+/-23.1 min. When the drug infusion was increased (0.69 microg kg(-1) min(-1)), each of five vessels remained patent. 3. Ex vivo platelet aggregation in response to arachidonic acid (AA) and ADP was examined in platelet rich plasma (PRP) prepared from citrate or heparin anticoagulated blood. CRL42796 reduced platelet reactivity at low and high doses in PRP from citrate anticoagulated blood. However, in PRP from heparin anticoagulated blood, only the higher infusion dose produced a significant reduction in ex vivo platelet responses. 4. A combination of oral aspirin (4.6 mg kg(-1) -41, -17 h) and the low infusion dose of CRL42796 did not produce an additional benefit beyond that provided by CRL42796 alone. 5. Coronary artery thrombosis was inhibited in four of five vessels treated with the lower infusion dose of CRL42796 and in five of five vessels treated with the higher infusion. Time to thrombosis increased with both doses (Control, 90.8+/-10.4 min; low dose, 165.8+/-14.2 min; high dose, >180.0+/-0 min). 6. The results indicate that CRL42796 is an effective in vivo antithrombotic agent against experimentally-induced carotid and coronary artery thrombosis.

C-reactive-protein-associated Increase in Myocardial Infarct Size After Ischemia/reperfusion

The Journal of Pharmacology and Experimental Therapeutics. Dec, 2002  |  Pubmed ID: 12438521

C-Reactive protein (CRP), a marker for acute inflammation, is associated with increased risk of cardiovascular events. The mechanism underlying this association is uncertain. An acute inflammatory response was induced in rabbits by subcutaneous injection of croton oil (CO) 1 to 3 days before 30 min of regional myocardial ischemia/180 min of reperfusion. CO treatment increased plasma CRP from below the limit of detection to 2.5 +/- 0.5 mg/dl and was associated with an increase in infarct size expressed as percentage of risk region [32 +/- 6% vehicle controls (n = 7) to 47 +/- 9% CO-treated rabbits (n = 7; P < 0.05]. After 10 min of ischemia and 180 min reperfusion, no infarct was found in controls; however, an infarct of 7 +/- 1% was found in CO-treated rabbits (P < 0.05; CRP, 2.3 +/- 0.4 mg/dl). The CRP-related increase in infarct size was not observed in croton oil-treated, C6-deficient rabbits (n = 5/group), indicating the involvement of complement. In these rabbits, infarct size was 22 +/- 2% (P < 0.05) despite having plasma CRP of 4.3 +/- 0.4 mg/dl. The CRP-associated increase in infarct size was ameliorated by pretreatment with heparin (n = 7; infarct size 33 +/- 3%; CRP, 2.3 +/- 0.3 mg/dl; P < 0.05) or N-acetylheparin (n = 7; infarct size 23 +/- 4%; CRP, 3.1 +/- 0.5 mg/dl; P < 0.05). These observations may explain why increased serum CRP is associated with an augmented risk for cardiovascular events.

Apoptosis in Myocardial Infarction

Annals of Medicine. 2002  |  Pubmed ID: 12523502

Apoptosis, one of the major forms of cell death, has been implicated in different cardiovascular diseases. In this paper we review many of the different studies that have been performed to address the occurrence of apoptotic cell death associated with myocardial infarction. A definitive differentiation between apoptosis and other forms of cell death is still needed, mainly because of differences and limitations of the methods used for detection. In myocardial infarction apoptosis has been reported at acute stages of evolution in the ischemic area as well as in remote zones. In the ischemic area it might be a determinant of the final size of the infarct and it seems to depend on the presence of post-ischemic reperfusion. However, the incidence of apoptosis reported until now varies widely. In the myocardium remote from the ischemic area it might be associated with the progression towards heart failure. At present, the role and significance of apoptosis in myocardial infarction is rather inconclusive. Further studies are needed to solve methodological uncertainties and clarify the mechanisms involved in the process of cell death, which is particularly important as a basis for therapeutic interventions.

SNC-80-induced Preconditioning: Selective Activation of the Mitochondrial Adenosine Triphosphate-gated Potassium Channel

Journal of Cardiovascular Pharmacology. May, 2003  |  Pubmed ID: 12717105

Pharmacologic preconditioning by delta-opioid agonists occurs via activation of an adenosine triphosphate (ATP)-gated potassium channel (I(KATP)). Opening of mitochondrial I(KATP) confers pharmacologic preconditioning whereas opening the sarcolemmal I(KATP) shortens action potential duration and is proarrhythmic. This study investigated whether SNC-80, a selective delta-opioid agonist, is associated with development of ventricular arrhythmia due to activation of I(KATP). Rabbit isolated hearts were subjected to 12 min of hypoxia and 40 min of reoxygenation after pretreatment with SNC-80 (1 microM, n = 6), pinacidil (1.25 microM, n = 12), or BMS-191095 (6.0 microM, n = 4). Nine additional hearts served as controls. The cytoprotective effects of SNC-80 at a concentration of 1 microM were confirmed using 30 min of regional ischemia followed by 120 min of reperfusion. Ventricular fibrillation (VF) developed in 11 of 12 pinacidil-treated hearts whereas none of the SNC-80-treated (zero of six) hearts developed VF (P < 0.001 compared with pinacidil pretreatment) and zero of four BMS-191095-pretreated hearts developed VF. Similarly, zero of nine control hearts developed VF. SNC-80 reduced infarct size expressed as a percentage of the area at risk from 33 +/- 4% to 14 +/- 3% (P = 0.004) compared with control. SNC-80, which selectively activates the delta-opioid receptor, provided cytoprotection but did not induce VF after hypoxia reoxygenation. The results indicate that pinacidil-induced nonselective activation of I(KATP) results in proarrhythmia that is dependent on activation of the sarcolemmal I(KATP). Selectivity for the mitochondrial I(KATP) is necessary to prevent induction of a proarrhythmic state.

Glycoprotein IIb/IIIa Receptor Antagonist (2S)-2-[(2-Naphthyl-sulfonyl)amino]-3-[[2-([4-(4-piperidinyl)-2-[2-(4-piperidinyl)ethyl] Butanoyl]amino)acetyl]amino]propanoic Acid Dihydrochloride (CRL42796), in Combination with Aspirin And/or Enoxaparin, Prevents Coronary Artery Rethrombosis After Successful Thrombolytic Treatment by Recombinant Tissue Plasminogen Activator

The Journal of Pharmacology and Experimental Therapeutics. Aug, 2003  |  Pubmed ID: 12734394

The antithrombotic effect of the glycoprotein IIb/IIIa (GPIIb/IIIa) antagonist (2S)-2-[(2-naphthyl-sulfonyl)amino]-3-[[2-([4-(4-piperidinyl)-2-[2-(4-piperidinyl)ethyl] butanoyl]amino)acetyl]amino] propanoic acid dihydrochloride (CRL42796), administered alone, or in combination with aspirin, and/or enoxaparin, was examined in a canine left circumflex (LCX) coronary artery rethrombosis model. The electrolytic induction of arterial thrombosis was followed by intracoronary recombinant tissue plasminogen activator administration to achieve thrombolysis, and the adjunctive therapy was initiated 15 min earlier and maintained for 4 h. Thirty-five purpose-bred beagle dogs were randomized to receive one of the following treatments: group 0 (n = 6, placebo); group 1 (n = 6, CRL42796 15 microg/kg i.v. loading dose followed by 0.31 microg/kg/min i.v. infusion), group 2 (n = 6, aspirin 7 mg/kg, administered orally, at -47, -23, -17 h before entry into the experimental protocol); group 3 (n = 6, aspirin + CRL42796); group 4 (n = 6, aspirin + enoxaparin 0.6 microg/kg i.v. loading dose followed by 6.0 microg/kg/min i.v. infusion); and group 5 (n = 5, aspirin + CRL42796 + enoxaparin). The incidence of LCX reocclusion was as follows: group 0, 6/6; group 1, 3/6; group 2, 5/6; group 3, 2/6; group 4, 2/6; and group 5, 0/5. Aspirin pretreatment increased the tongue-bleeding time, whereas the addition of CRL42796 or enoxaparin did not prolong bleeding time to a further degree. However, the combination of the three drugs did increase bleeding time significantly, from 173.9 +/- 19.8 to 620.0 +/- 98.7 s. In conclusion, low-dose CRL42796 together with aspirin and enoxaparin prevented coronary artery rethrombosis, although bleeding time was prolonged. The latter may be of concern in the clinical use of combination therapy.

Prevention of Carotid Artery Thrombosis After Oral Administration of the Glycoprotein IIb/IIIa Antagonist CRL42796

Journal of Cardiovascular Pharmacology. Jul, 2003  |  Pubmed ID: 12827029

This study investigates the effect of the glycoprotein IIb/IIIa receptor antagonist CRL42796 in a canine model of carotid artery thrombosis. Both carotid arteries developed occlusive thrombosis in each of the five control animals (time to occlusion: right carotid artery, 92.6 minutes; left carotid artery, 89.0 minutes). A single oral dose of CRL42796 (3 mg/kg) prevented occlusive thrombosis in 4 of 6 vessels and increased time to thrombosis, albeit not significantly (right carotid artery, 134.1 minutes; left carotid artery, 145.0 minutes). When the initial dose of CRL42796 was followed by a second oral dose (3 mg/kg) 2 hours later, 10 of 10 carotid arteries remained patent throughout the period of electrolytic injury. CRL42796 reduced thrombus weight in both treatment protocols. Ex vivo platelet aggregation with arachidonic acid (AA) or adenosine diphosphate (ADP) was reduced at 120, 240, and 360 minutes after two doses of CRL42796. A single oral dose reduced ADP-induced responses at 240 and 360 minutes, but significant effects were not observed with AA. Bleeding time increased 360 minutes after two oral doses of CRL42796, but not at 120 minutes. Bleeding time was unchanged with the single dose of CRL42796. The results demonstrate that oral administration of CRL42796 prevents carotid artery thrombosis in response to deep vessel wall injury and may have potential value to be characterized in extended preclinical and clinical study.

17Beta-estradiol As a Receptor-mediated Cardioprotective Agent

The Journal of Pharmacology and Experimental Therapeutics. Oct, 2003  |  Pubmed ID: 12893838

Cardiac tissue that undergoes an ischemic episode exhibits irreversible alterations that become more extensive upon reperfusion. Estrogen treatment has been reported to protect against reperfusion injury, but the mechanism remains unknown. The cardioprotective effects of 17beta-estradiol, a biologically active form of the hormone, and 17alpha-estradiol were assessed in an in vivo occlusion-reperfusion model. Anesthetized, ovariectomized rabbits were administered 17beta-estradiol (20 microg), 17alpha-estradiol (1 mg), or vehicle intravenously 30 min before a 30-min occlusion of the left anterior descending (LAD) coronary artery followed by 4 h of reperfusion. Infarct size as a percentage of area at risk decreased in the 17beta-estradiol-treated group (18.8 +/- 1.7) compared with 17alpha-estradiol (41.9 +/- 4.8; P < 0.01) or vehicle groups (48 +/- 5.5; P < 0.001). Similar results were obtained when infarct size was expressed as a percentage of total left ventricle. The second objective of the study was to assess fulvestrant (Faslodex, ICI 182,780), an estrogen receptor antagonist, for its effects on infarct size in ovariectomized female rabbits treated with 17beta-estradiol. ICI 182,780 was administered intravenously 1 h before the administration of 17beta-estradiol (20 microg) or vehicle. The hearts were subjected to 30-min LAD coronary artery occlusion and 4 h of reperfusion. Pretreatment with ICI 182,780 significantly limited the infarct size sparing effect of 17beta-estradiol when expressed as a percentage of the risk region (53.0 +/- 5.0). The results indicate that 17beta-estradiol protects the heart against ischemia-reperfusion injury and that the observed cardioprotection is mediated by the estrogen receptor.

Risk of Ventricular Proarrhythmia with Selective Opening of the Myocardial Sarcolemmal Versus Mitochondrial ATP-gated Potassium Channel

The Journal of Pharmacology and Experimental Therapeutics. May, 2004  |  Pubmed ID: 14747611

Myocardial ATP-gated potassium channels (K-ATPs) are critical in the intracellular signaling cascade resulting in ischemic preconditioning (IP). Mitochondrial K-ATP channels seem to be responsible for IP, whereas the functions of K-ATP channels in the sarcolemmal membrane are less well understood. The proarrhythmic potential of specific versus nonspecific opening of K-ATP channels has not been investigated. In this study, Langendorff-perfused rabbit hearts were exposed to either pinacidil (1.25 microM), a nonselective K-ATP channel agonist, or selective mitochondrial or sarcolemmal K-ATP channel agonists or antagonists. The hearts were then subjected to 12 min of hypoxic perfusion and 40 min of reoxygenation. Hearts were monitored for the induction of ventricular fibrillation (VF). No heart subjected to hypoxia-reoxygenation without drug treatment developed VF (0 of 5). Pinacidil pretreatment induced VF (12 of 14; p = 0.004 versus control). Pinacidil's effect was blocked by HMR-1098 (1-[5-[2-(5-chloro-o-anisamide)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea) (1 microM), a selective sarcolemmal K-ATP channel antagonist (1 of 7; p = 0.007 versus pinacidil; N.S. versus control). Hearts pretreated with 5-hydroxydecanoate (5-HD) (100 microM), a putatively selective mitochondrial K-ATP channel blocker developed VF in one of eight trials (N.S. versus control). 5-HD did not alter the effects of pinacidil (6 of 8; p < 0.05 versus control; N.S. versus pinacidil alone). Selective mitochondrial K-ATP channel activation with [(3R)-trans-4-((4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)dimethyl-2H-1-benzopyran-6-carbonitril monohydrochloride] (BMS-191095) (6 microM) resulted in zero of five hearts developing VF (N.S. versus control). Our data suggest that selective opening of the sarcolemmal K-ATP channel during hypoxia-reoxygenation induced VF, whereas opening of the mitochondrial channel was not associated with VF. The findings suggest that caution should be exercised when developing compounds aimed at inducing IP, and nonspecific opening of the K-ATP channel should be avoided.

The Preparation and Characterization of Novel Peptide Antagonists to Thrombin and Factor VIIa and Activation of Protease-activated Receptor 1

The Journal of Pharmacology and Experimental Therapeutics. Nov, 2004  |  Pubmed ID: 15210836

Thrombin and protease-activated receptor 1 (PAR1) activation antagonists were prepared based upon the peptide RPPGF, the angiotensin-converting enzyme breakdown product of bradykinin. A library of 72 peptides consisting of d and/or synthetic amino acids was designed with various substitutions in positions 1 to 5 in Arg-Pro-Pro-Gly-Phe (RPPGF). Two compounds, rOicPGF (TH146) and betaAK2K-4(rOicPGF) (MAP4-TH146), were characterized further. TH146 or MAP4-TH146 completely inhibits threshold gamma-thrombin-induced platelet aggregation at a concentration of 142 +/- 0.05 or 19 +/- 0.06 microM, respectively. TH146 completely inhibits threshold alpha-thrombin-induced washed platelet aggregation at 444 +/- 0.04 microM. TH146 or MAP4-TH146 blocks 2 nM alpha-thrombin-induced fibroblast calcium mobilization with an IC(50) value of 110 or 18 microM, respectively. Furthermore, significant prolongation of the activated partial thromboplastin time, prothrombin time, or thrombin clotting time occurs at 31, 62, or 7.8 microM TH146 and 0.4, 6.25, or 1.56 microM MAP4-TH146, respectively. TH146 and MAP4-TH146 inhibit both alpha-thrombin with a K(i) value of 97 and 49 microM, respectively, and factor VIIa with a K(i) value of 44 and 5 microM, respectively. Both TH146 and MAP4-TH146 specifically bind to the exodomain of recombinant PAR1. MAP4-TH146 (200 microM) completely blocks thrombocytin, a PAR1-activating snake venom protease, without inhibiting the enzyme's active site. TH146 inhibits gamma-thrombin-induced aggregation of mouse platelets, prolongs mouse bleeding times, and delays the time to mouse carotid artery thrombosis. TH146 and MAP4-TH146 inhibit human and mouse platelet aggregation and mouse thrombosis. Analogs of RPPGF are model compounds to develop PAR1 activation antagonists as well as direct inhibitors to thrombin and factor VIIa.

Apolipoprotein A-IMilano and 1-palmitoyl-2-oleoyl Phosphatidylcholine Complex (ETC-216) Protects the in Vivo Rabbit Heart from Regional Ischemia-reperfusion Injury

The Journal of Pharmacology and Experimental Therapeutics. Dec, 2004  |  Pubmed ID: 15375174

Ex vivo studies demonstrated that a synthetic high-density lipoprotein (HDL) comprised of a complex of recombinant apolipoprotein A-IMilano and 1-palmitoyl-2-oleoyl phosphatidylcholine protects the isolated rabbit heart from reperfusion injury. Therefore, we sought to determine whether a pharmaceutical preparation of this complex, ETC-216, was cardioprotective in an in vivo model of left anterior descending artery (LAD) occlusion and reperfusion. Initially, ETC-216 (100 mg/kg) was tested in acute (one-treatment) and chronic (two-treatment) i.v. administrations. ETC-216-treated rabbits developed smaller infarcts expressed as percentage of area at risk (p <0.01) compared with vehicle treatments. No differences were noted between chronic and acute administration. Therefore, ETC-216 (10, 3, or 1 mg/kg) or equivalent vehicle volumes were acutely infused. Compared with vehicle, ETC-216 reduced infarct size as a percentage of the area at risk at 10 (p <0.0005) and 3 mg/kg (p <0.05). No significant differences occurred at 1 mg/kg. To determine whether ETC-216 could protect the heart after initiation of ischemia, the synthetic HDL (10 mg/kg) was infused intravenously beginning 5 min before the end of 30 min of LAD occlusion. Infarct size as percentage of the area at risk was 31.6 +/- 3.0 (ETC-216) versus 49.5 +/- 2.5 (vehicle) (p <0.001), and as percentage of left ventricle was 19.7 +/- 1.6 (ETC-216) versus 34.1 +/- 2.3 (vehicle) (p <0.0005). Electron microscopy demonstrated that ETC-216 prevented irreversible cardiac damage as assessed by mitochondrial granulation and sarcomere contraction band formation. These findings suggest ETC-216 reduces reperfusion injury and may have utility for coronary artery revascularization procedures.

Dermatan Disulfate (Intimatan) Prevents Complement-mediated Myocardial Injury in the Human-plasma-perfused Rabbit Heart

International Immunopharmacology. Feb, 2005  |  Pubmed ID: 15652767

Intimatan (dermatan 4,6-O-disulfate), a heparin cofactor II agonist, is a highly sulfated negatively charged semisynthetic polysaccharide. The present study examined the hypothesis that Intimatan reduces complement-mediated myocardial injury. The rabbit isolated heart was perfused with 4% normal human plasma (NHP) as a source of complement in the absence or presence of Intimatan (5 microM). Heat-inactivated human plasma (HIHP) was used as a negative control. Previous studies demonstrated that contact of rabbit tissue with human plasma results in activation of the alternative pathway of the human complement system, leading to irreversible myocardial injury. In the presence of NHP, left ventricular end-diastolic pressure (LVEDP) was increased significantly to 61.8+/-11.7 mm Hg compared to a value of 17.2+/-6.1 mm Hg in hearts perfused in the presence of HIHP. Left ventricular developed pressure (LVDP) was reduced significantly upon exposure to NHP, 19.3+/-10.2 (NHP) vs. 54.0+/-8.0 mm Hg (HIHP). Functional impairment in the presence of NHP was accompanied by a significant release of cardiac troponin I (cTnI; 131.8+/-20.3 ng/ml) as compared to hearts exposed to HIHP (0.8+/-0.8). Intimatan treatment improved cardiac function and maintained viability of cardiac myocytes (LVEDP 14.6+/-5.6, LVDP 58.0+/-8.1 mm Hg and cTnI 6.7+/-5.2 ng/ml). Immunohistochemical staining demonstrated that Intimatan pretreatment prevented deposition of the human membrane attack complex (MAC) in hearts exposed to NHP. The results indicate that Intimatan, a glycosaminoglycan (GAG), can reduce tissue injury and preserve organ function that otherwise would be compromised during activation of the human complement cascade.

Ischemia Depletes Dystrophin and Inhibits Protein Synthesis in the Canine Heart: Mechanisms of Myocardial Ischemic Injury

Journal of Molecular and Cellular Cardiology. May, 2005  |  Pubmed ID: 15850566

In this study we tested our previous hypothesis that ischemia is a multifactorial injurious event involving all components of the myocyte simultaneously. This hypothesis was based on ultrastructural findings and was now tested again by protein analysis of sarcolemmal structural proteins and of markers of transcriptional and translational activities. This knowledge may help to clarify the cellular mechanisms involved in progression of acute ischemic myocardial injury and reperfusion. Therefore, we investigated all three intracellular/extracellular linkage systems of the sarcolemma using antibodies against dystrophin, beta-dystroglycan, gamma-sarcoglycan, vinculin, beta1-integrin, laminin, and spectrin. In addition, antibodies were used to evaluate membrane permeability (albumin), transcriptional efficacy (non-snRNP splicing factor SC-35), and translational capacity (phosphorylated p70 ribosomal protein S6 kinase). Tissue samples were obtained from a canine model of regional myocardial ischemia (90 min or 4.5 h) with or without reperfusion. Immunoconfocal microscopy and Western blotting revealed that the rank order of sensitivity was the following: dystrophin, beta-dystroglycan, gamma-sarcoglycan, vinculin, spectrin, integrin and laminin. Different levels of dystrophin loss indicate reversible/irreversible injury as established by albumin uptake and electron microscopy. Dystrophin depletion closely coincided with generally depressed transcription and translation. These changes occurred simultaneously in a time-dependent manner and persisted during reperfusion. In conclusion, damage of the different structural proteins results in membrane destabilization and disruption of the contractile apparatus from the sarcolemma. These changes, concomitantly associated with disturbances in transcription and translation, are major mechanisms determining the transition to irreversibility of myocardial ischemic injury and confirm our hypothesis that ischemia is a multifactorial injurious event involving all components of the cardiac myocyte.

Disodium Disuccinate Astaxanthin (Cardax) Attenuates Complement Activation and Reduces Myocardial Injury Following Ischemia/reperfusion

The Journal of Pharmacology and Experimental Therapeutics. Aug, 2005  |  Pubmed ID: 15872041

Carotenoids are a naturally occurring group of compounds that possess antioxidant properties. Most natural carotenoids display poor aqueous solubility and tend to form aggregates in solution. Disodium disuccinate astaxanthin (DDA; Cardax) is a water-dispersible synthetic carotenoid that rapidly and preferentially associates with serum albumin, thereby preventing the formation of supramolecular complexes and facilitating its efficacy after parenteral administration. This study investigated the ability of DDA to reduce inflammation and myocardial injury in a rabbit model of ischemia/reperfusion. DDA (50 mg/kg/day) or saline was administered i.v. for 4 consecutive days before the initiation of the protocol for induction of myocardial ischemia/reperfusion. On the 5th day, rabbits underwent 30 min of coronary artery occlusion, followed by a 3-h reperfusion period. Myocardial infarct size, as a percentage of the area at risk, was calculated for both groups. Infarct size was 52.5 +/- 7.5% in the vehicle-treated (n = 9) and 25.8 +/- 4.7% in the DDA-treated (n = 9) animals (p < 0.01 versus vehicle; mean myocardial salvage = 51%). To evaluate the anti-inflammatory effects of DDA, complement activity was assessed at the end of reperfusion using a red blood cell lysis assay. DDA administration significantly reduced (p < 0.01) the activation of the complement system in the serum. The current results, coupled with the well established antioxidant ability of carotenoids, suggest that the mechanism(s) of action by which DDA reduces the tissue damage associated with reperfusion injury may include both antioxidant and anticomplement components.

Sulodexide Attenuates Myocardial Ischemia/reperfusion Injury and the Deposition of C-reactive Protein in Areas of Infarction Without Affecting Hemostasis

The Journal of Pharmacology and Experimental Therapeutics. Feb, 2005  |  Pubmed ID: 15365091

Several glycosaminoglycans (GAGs) have been demonstrated to protect the ischemic heart against reperfusion injury, in part, by modulating activation of the complement cascade. The present study assessed the cardioprotective effects of sulodexide (KRX-101), a mixture of GAGs composed of 80% low-molecular mass heparin and 20% dermatan sulfate. KRX-101 differs from other GAGs (e.g., heparin) in that it has limited anticoagulant efficacy and can be administered orally. The experimental protocol was designed to determine whether KRX-101 could protect the ischemic myocardium. Anesthetized New Zealand white rabbits underwent 30 min of coronary artery occlusion. Intravenous doses of KRX-101 (0.5 mg/kg, n = 10) or drug diluent (n = 10) were administered at the end of regional ischemia and at each hour of reperfusion. Infarct size, as a percentage of the area at risk, was calculated for both groups. Myocardial infarct size was 31.3 +/- 4.1% in the vehicle- and 17.3 +/- 3.2% in the KRX-101-treated animals (p < 0.05 versus vehicle). Activated partial thromboplastin times determined at baseline (preischemia) and at each hour of reperfusion (n = 4) were not significantly different between vehicle- and KRX-101-treated groups (p = N.S.). Myocardial injury was further assessed by measuring serum levels of cardiac-specific troponin I. KRX-101 administration significantly reduced (p < 0.05) the serum concentration of troponin I during reperfusion. The results suggest that KRX-101 may be an effective adjunctive agent in myocardial revascularization procedures, without the risk of increased bleeding.

Activation of Estrogen Receptor-alpha Protects the in Vivo Rabbit Heart from Ischemia-reperfusion Injury

American Journal of Physiology. Heart and Circulatory Physiology. Nov, 2005  |  Pubmed ID: 15994857

The estrogen receptor (ER) mediates estrogenic activity in a variety of organs, including those in the reproductive, cardiovascular, immune, and central nervous systems. Experimental studies have demonstrated that 17beta-estradiol (E2) protects the heart from ischemia-reperfusion injury. Two estrogen receptors, ER alpha and ER beta, mediate the actions of estrogen; however, it is not certain which ER mediates the cardioprotective effects of E2. In the present study, the ER-selective agonists 4,4',4''-[4-propyl-(1H)-pyrazole-1,3,5-triyl]tris-phenol (PPT; ER alpha) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; ER beta) were assessed for their cardioprotective potential in an in vivo rabbit model of ischemia-reperfusion injury. Anesthetized female rabbits were administered PPT (3 mg/kg), DPN (3 mg/kg), E2 (20 microg/rabbit), or vehicle intravenously 30 min before a 30-min occlusion of the left anterior descending coronary artery followed by 4 h of reperfusion. Acute treatment with E2 (17.7 +/- 2.9%; P < 0.001) and PPT (18.1 +/- 2.9%; P < 0.001), but not DPN (45.3 +/- 2.4%) significantly decreased infarct size as a percent of area at risk compared with vehicle (45.3 +/- 2.4%). Coadministration of PPT or E2 with the ER antagonist ICI-182,780 limited the infarct size-sparing effect of the compounds (43.8 +/- 6.6% and 40.6 +/- 5.7% respectively, expressed as a percentage of risk region). PPT reduced the release of cardiac-specific troponin-I and reduced the tissue deposition of the membrane attack complex and C-reactive protein similar to that of E2. The results indicate that activation of ER alpha, but not ER beta, is required for the observed cardioprotective effects of E2.

The Antithrombotic Effect of Melagatran in Combination with Clopidogrel And/or Aspirin (carotid Artery Primary Thrombosis Study)

Journal of Cardiovascular Pharmacology. Oct, 2005  |  Pubmed ID: 16160608

Melagatran with aspirin and/or clopidogrel was evaluated for prevention of arterial thrombosis in a model of vessel wall injury. Thirty-five dogs were randomized to receive placebo (n=14), aspirin (7 to 8 mg/kg, p.o. q12 h for three doses with the last dose administered 12 hours before surgery, n=7), clopidogrel (1 mg/kg p.o. QDx3, n=7), or aspirin+clopidogrel (n=7). The right carotid artery (RCA) was the control vessel, whereas the left carotid artery (LCA) was subjected to injury after administration of Melagatran (0.033 mg/kg i.v.+0.1 mg/kg/h). Clopidogrel, but not aspirin pretreatment, increased time (135.6+/-13.5 vs. 116.1+/-27.8 minutes) to RCA thrombosis versus placebo (88.1+/-10.5 minutes). Melagatran prolonged time to occlusion (min) in the LCA (192.4+/-10.9) versus the placebo-treated RCA (88.1+/-10.5). Addition of Melagatran plus aspirin or clopidogrel prevented formation of occlusive thrombosis, in all LCAs. A two-fold increase in tongue bleeding time was observed after aspirin+Melagatran (178.6+/-14.7 to 347.1+/-87.3 seconds) or clopidogrel+Melagatran (279.9+/-97.3 to 437.1+/-142.5 seconds). However, the combination of aspirin and clopidogrel prevented occlusive thrombosis in the RCA and the subsequent addition of Melagatran did not further increase bleeding time. The combination of Melagatran+aspirin or clopidogrel can reduce formation of occlusive arterial thrombosis without eliciting a significant increase in bleeding-time.

Effect of Thrombolysis on Myocardial Injury: Recombinant Tissue Plasminogen Activator Vs. Alfimeprase

American Journal of Physiology. Heart and Circulatory Physiology. Mar, 2006  |  Pubmed ID: 16243915

Plasmin-dependent thrombolytic agents are potentially prothrombotic and proinflammatory. Alfimeprase, a zinc-containing metalloproteinase, degrades fibrin directly and achieves thrombolysis independent of plasmin formation. This study examines the hypothesis that thrombolysis in the absence of plasmin generation results in improved myocardial salvage on reperfusion. The thrombolytic effects of recombinant tissue plasminogen activator [rt-PA; 0.022 mg/kg, 1/10 of which was administered as a loading dose; the rest (9/10) was infused over 60 min by intracoronary (ic) administration] or alfimeprase (0.5 mg/kg over 1 min ic) were evaluated in a canine model of arterial thrombosis involving electrolytic injury of the left circumflex (LCX) coronary artery. Both agents induced thrombolysis, with onset of reperfusion being more rapid after alfimeprase compared with rt-PA (1.5 +/- 0.6 vs. 10.1 +/- 2.1 min). In the absence of adjunctive therapy, time to reocclusion after alfimeprase was 3.2 +/- 0.5 min compared with 77.5 +/- 31.9 min with rt-PA. The glycoprotein IIb/IIIa platelet receptor antagonist CRL-42796 prolonged reperfusion time after thrombolysis with alfimeprase or rt-PA. The effect of each lytic agent on myocardial infarct size was examined in a separate group of dogs subjected to 60 min of LCX coronary artery ligation and 4 h of reperfusion. Myocardial infarct size, expressed as percentage of the risk region, was larger (32.16 +/- 3.95%) after rt-PA compared with alfimeprase (19.85 +/- 3.61%) or that of the saline control group (18.46 +/- 3.34%). rt-PA in contrast to alfimeprase, a direct-acting fibrinolytic agent, is associated with an increase in myocyte reperfusion injury.

Microvascular Permeabilization and Cardiomyocyte Injury Provoked by Myocardial Contrast Echocardiography in a Canine Model

Journal of the American College of Cardiology. Apr, 2006  |  Pubmed ID: 16580537

The aim of this research was to evaluate the potential for myocardial contrast echocardiography (MCE) to provoke microscale bioeffects in a canine model.

Effect of Sodium/hydrogen Exchange Inhibition on Myocardial Infarct Size After Coronary Artery Thrombosis and Thrombolysis

Pharmacology. 2006  |  Pubmed ID: 16899991

This study examines the cardioprotective effects of Na+/H+ exchange inhibition with BIIB-722 or ischemic preconditioning after occlusive thrombus formation and subsequent thrombolysis for reperfusion. Coronary artery thrombosis was induced by vessel wall electrolytic injury. Thrombotic occlusion was maintained for 60 or 90 min in 4 different groups: (1) control; (2) Na+/H+ exchange inhibitor, BIIB-722 (3 mg/kg) before occlusion; (3) BIIB-722 (0.75 mg/kg) before reperfusion; (4) ischemic preconditioning (4 x 5 min). Thrombolysis with intracoronary recombinant tissue plasminogen activator produced reperfusion in 6.3 +/- 1.4 min (average for 68 dogs). After restoration of blood flow, vessel patency was maintained for 4 h with the glycoprotein IIb/IIIa receptor antagonist, BIBU 52ZW. BIIB-722, administered before (26.9 +/- 3.6%) or after (22.0 +/- 2.3%) 60-min ischemia or preconditioning (18.4 +/- 2.8%), produced comparable and significant reductions in infarct size (percent of area at risk) compared to controls (47.2 +/- 2.0%). After 90 min of ischemia, BIIB-722 administered before occlusion (37.3 +/- 1.1%) and ischemic preconditioning (35.0 +/- 4.8%) provided significant cardioprotection compared to control (45.9 +/- 1.8%). BIIB-722 was not cardioprotective when administered during occlusion (48.0 +/- 2.4%). The results indicate that Na+/H+ exchange inhibition and preconditioning provide a comparable degree of cardioprotection against 60 min of regional ischemia. However, when the regional ischemic period is extended to 90 min, the degree of cardioprotection is markedly reduced. Further studies incorporating clinically relevant events such as thrombosis and thrombolysis are required before one can conclude that Na+/H+ exchange inhibition is effective against more prolonged myocardial ischemia.

Preclinical Evaluation of S18886 in an Experimental Model of Coronary Arterial Thrombosis

Journal of Cardiovascular Pharmacology. Nov, 2006  |  Pubmed ID: 17110806

The specific thromboxane receptor antagonist, S18886, was evaluated for prevention of coronary arterial thrombosis and myocardial ischemia-reperfusion in anesthetized canines. For the primary thrombosis study in left circumflex (LCX) coronary artery, 26 dogs were randomized to receive either vehicle (n = 7) or intravenous S18886 (0.3 mg/kg, n = 6; 1.0 mg/kg, n = 6; and 3.0 mg/kg, n = 7). The respective times to occlusion after S18886 were as follows: 56.8 +/- 9.3, 83.5 +/- 14.9, and 92.4 +/- 15.7 minutes compared to 43.3 +/- 8.2 minutes after vehicle. S18886 caused a minimal increase in tongue bleeding time and a significant decrease in ex vivo platelet aggregation to arachidonic acid or U46619. Another 37 dogs were randomized to receive placebo (n = 12), clopidogrel 1.0 mg/kg p.o. QDX3 (n = 9), clopidogrel + S18886 0.3 (n = 9) or 1.0 (n = 7) mg/kg intravenous. Clopidogrel produced a 50% reduction in adenosine diphosphate-induced platelet aggregation and a slight increase in the time to occlusion. However, clopidogrel + S18886 1.0 mg/kg prevented occlusive thrombus formation in most of the coronary vessels over 6 hours. S18886 did not alter myocardial infarct size in the ischemia-reperfusion model. In conclusion, S18886 alone caused a dose-dependent prolongation in the time to primary occlusive coronary artery thrombosis, whereas S18886 + clopidogrel displayed effective in preventing occlusive thrombus formation with only a moderate increase of tongue-bleeding time.

Sulodexide: a Renewed Interest in This Glycosaminoglycan

Cardiovascular Drug Reviews. Fall-Winter, 2006  |  Pubmed ID: 17214598

Glycosaminoglycans (GAGs) are the most abundant group of heteropolysaccharides found in the body. These long unbranched molecules contain a repeating disaccharide unit. GAGs are located primarily in the extracellular matrix or on the surface of cells. These molecules serve as lubricants in the joints while at the same time providing structural rigidity to cells. Sulodexide is a highly purified glycosaminoglycan composed of a fast mobility heparin fraction as well as dermatan sulfate. Sulodexide differs from other glycosaminoglycans, like heparin, by having a longer half-life and a reduced effect on systemic clotting and bleeding. In addition, sulodexide demonstrates a lipolytic activity that is increased in comparison to heparin. Oral administration of sulodexide results in the release of tissue plasminogen activator and an increase in fibrinolytic activities. An increasing body of research has demonstrated the safety and efficacy of sulodexide in a wide range of vascular pathologies.

Intimatan (dermatan 4,6-O-disulfate) Prevents Rethrombosis After Successful Thrombolysis in the Canine Model of Deep Vessel Wall Injury

Thrombosis Research. 2006  |  Pubmed ID: 15893368

Intimatan (dermatan 4,6-O-disulfate), a heparin cofactor II (HCII) agonist, inhibits both the fluid phase and thrombus bound thrombin. The efficacy of Intimatan as an adjunctive anticoagulant during thrombolysis was evaluated in the canine model of arterial injury.

Medroxyprogesterone Acetate Prevents the Cardioprotective and Anti-inflammatory Effects of 17beta-estradiol in an in Vivo Model of Myocardial Ischemia and Reperfusion

American Journal of Physiology. Heart and Circulatory Physiology. Sep, 2007  |  Pubmed ID: 17434982

Previous studies demonstrated the protective effects of estrogen administration in models of cardiovascular disease. However, there is a discrepancy between these data and those from the recent clinical trials with hormone replacement therapy in menopausal women. One possible explanation for the divergent results is the addition of progestin to the hormone regimen in the Women's Health Initiative and the Heart and Estrogen/Progestin Replacement Study trials. The aim of the present study was to examine the effects of a combination of 17beta-estradiol (E(2), 20 microg) and medroxyprogesterone acetate (MPA, 80 microg) on infarct size in New Zealand White rabbits. Infarct size as a percentage of the area at risk was significantly reduced by administration of E(2) 30 min before induction of myocardial ischemia compared with vehicle (19.5 +/- 3.1 vs. 55.7 +/- 2.6%, P < 0.001). However, E(2) + MPA failed to elicit a reduction in infarct size (52.5 +/- 4.6%), and MPA had no effect (50.8 +/- 2.6%). E(2) also reduced serum levels of cardiac troponin I, immune complex deposition in myocardial tissue, activation of the complement system, and lipid peroxidation. All these effects were reversed by MPA. The results suggest that MPA antagonizes the infarct-sparing effects of E(2), possibly through modulation of the immune response after ischemia and reperfusion.

The Pathway-selective Estrogen Receptor Ligand WAY-169916 Reduces Infarct Size After Myocardial Ischemia and Reperfusion by an Estrogen Receptor Dependent Mechanism

Journal of Cardiovascular Pharmacology. Jun, 2007  |  Pubmed ID: 17577105

Previous studies have shown that estrogen treatment protects the heart from reperfusion injury. The adverse effects of long-term estrogen treatment limit its clinical use and emphasize the need for the development of specific pharmacological interventions such as pathway-selective estrogen receptor (ER) ligands. Pathway-selective ER ligands are compounds that retain estrogen's anti-inflammatory ability, but they are devoid of conventional estrogenic action. In the present study, the pathway-selective ER ligand WAY-169916 was assessed for its cardioprotective potential in an in vivo model of ischemia-reperfusion injury. Anesthetized, ovariectomized rabbits were administered WAY-169916 (1 mg/kg), 17beta-estradiol (E2; 20 microg/rabbit), or vehicle intravenously 30 minutes before a 30-minute occlusion and 4 hours of reperfusion. Acute treatment with either WAY-169916 or E2 resulted in a decrease in infarct size, expressed as a percent of area at risk (WAY-169916, 21.2 +/- 3.3; P < 0.001 and E2, 18.8 +/- 1.7; P < 0.001) compared with vehicle 59.4 +/- 5.4). Pretreatment with estrogen receptor antagonist ICI 182,780 significantly limited the infarct size sparing effect of both WAY-169916 and E2 when expressed as a percent of the risk region (WAY 169916, 47.4 +/- 4.4; E2, 53.01 +/- 5.0). The results demonstrate that WAY-169916 protects the heart against ischemia-reperfusion injury through an ER-dependent mechanism.

Effects of Cyclooxygenase Inhibition on Canine Coronary Artery Blood Flow and Thrombosis

American Journal of Physiology. Heart and Circulatory Physiology. Jan, 2008  |  Pubmed ID: 17921331

This study was designed to determine the effect of inhibitors of cyclooxygenase (COX)-1, COX-2, and the nonselective COX inhibitor naproxen on coronary vasoactivity and thrombogenicity under baseline and lipopolysaccharide (LPS)-induced inflammatory conditions. We hypothesize that endothelial COX-1 is the primary COX isoform in the canine normal coronary artery, which mediates arachidonic acid (AA)-induced vasodilatation. However, COX-2 can be induced and overexpressed by inflammatory mediators and becomes the major local COX isoform responsible for the production of antithrombotic prostaglandins during systemic inflammation. The interventions included the selective COX-1 inhibitor SC-560 (0.3 mg/kg iv), the selective COX-2 inhibitor nimesulide (5 mg/kg iv), or the nonselective COX inhibitor naproxen (3 mg/kg iv). The selective prostacyclin (IP) receptor antagonist RO-3244794 (RO) was used as an investigational tool to delineate the role of prostacyclin (PGI(2)) in modulating vascular reactivity. AA-induced vasodilatation of the left circumflex coronary artery was suppressed to a similar extent by each of the COX inhibitors and RO. The data suggest that AA-induced vasodilatation in the normal coronary artery is mediated by a single COX isoform, the constitutive endothelial COX-1, which is reported to be susceptible to COX-2 inhibitors. The effect of the COX inhibitors on thrombus formation was evaluated in a model of carotid artery thrombosis secondary to electrolytic-induced vessel wall injury. Pretreatment with LPS (0.5 mg/kg iv) induced a systemic inflammatory response and prolonged the time-to-occlusive thrombus formation, which was reduced in the LPS-treated animals by the administration of nimesulide. In contrast, neither SC-560 nor naproxen influenced the time to thrombosis in the animals pretreated with LPS. The data are of significance in view of reported adverse cardiovascular events observed in clinical trials involving the use of selective COX-2 inhibitors, thereby suggesting that the endothelial constitutive COX-1 and the inducible vascular COX-2 serve important functions in maintaining vascular homeostasis.

Apolipoprotein A-IMilano/POPC Complex Attenuates Post-ischemic Ventricular Dysfunction in the Isolated Rabbit Heart

Atherosclerosis. Apr, 2008  |  Pubmed ID: 17945238

Irreversible myocardial injury is a potential consequence of coronary artery revascularization. Reperfusion leads to the production of oxidized products that can damage myocardium. High-density lipoproteins (HDL) are effective at removing oxidized lipids. We hypothesized that a synthetic HDL preparation, comprising recombinant apolipoprotein A-I(Milano) (apoA-I(M)) complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) (apoA-I(M)/POPC) would protect the heart from reperfusion injury. The ex vivo model consisted of rabbit hearts perfused by the Langendorff method. Hearts were equilibrated with Krebs-Henseleit buffer (10 min), pretreated with either apoA-I(M)/POPC (0.45 mg/mL) or vehicle (10 min), subjected to global ischemia (30 min) and reperfused for 60 min. ApoA-I(M)/POPC (n=7) prevented the left ventricular end-diastolic pressure elevation observed in the vehicle group (n=6) at the end of reperfusion (p<0.05). During reperfusion, coronary artery perfusion pressure increased in the controls (p<0.001), but not with apoA-I(M)/POPC. ApoA-I(M)/POPC reduced the release of creatine kinase at the end of the ischemic period (p<0.001). It also reduced cardiac left ventricle muscle lipid hydroperoxides by 46% (p<0.05). Direct comparison of the antioxidant potential indicated that recombinant apoA-I(M) was much more potent than apoA-I in attenuating low-density lipoprotein oxidation. Electron microscopy showed that apoA-I(M)/POPC prevented mitochondrial granulation, disorganization and sarcomere contraction band formation indicative of reperfusion injury. The apoA-I(M)/POPC complex thus appears to reduce reperfusion injury under global ischemic conditions, and may therefore have therapeutic application in the reduction of myocardial ischemia.

Disodium Disuccinate Astaxanthin Prevents Carotid Artery Rethrombosis and Ex Vivo Platelet Activation

Pharmacology. 2008  |  Pubmed ID: 18477858

The disodium disuccinate derivative of astaxanthin (DDA) is a carotenoid antioxidant under development for the treatment of ischemic cardiovascular events. Recent evidence suggests that reactive oxygen species (ROS) play an important role in platelet activation. This study seeks to investigate the effects of a reactive oxygen species quencher, DDA, in a canine model of carotid artery thrombosis.

Estrogen-mediated Protection in Myocardial Ischemia-reperfusion Injury

Cardiovascular Toxicology. 2008  |  Pubmed ID: 18683081

Before menopause, a woman has a relatively low risk for developing cardiovascular disease. After menopause, however, the risk increases nearly twofold and cardiovascular disease remains the number one cause of death among women. Observational trials and studies in animal models of cardiovascular disease suggested that females have reduced injury after myocardial ischemia and reperfusion injury. However, two large clinical trials, the women's health initiative (WHI) and the heart estrogen and progestin replacement study (HERS), found an increase in cardiovascular incidences in women taking hormone replacement therapy. The discrepancy between these data highlights the need for further research on the mechanism of estrogen in the cardiovascular system. Animal studies have demonstrated protective effects by endogenous estrogen (gender differences) and also by the administration of exogenous estrogen. In vivo studies suggest a possible anti-inflammatory mechanism of estrogen. Exogenous estrogen has been shown to have anti-oxidant activities. Pre-treatment with estrogen prior to myocardial ischemia and reperfusion causes a decrease in neutrophil infiltration into the irreversibly injured myocardium, decrease in C-reactive protein expression, and deposition of the membrane attack complex. This review will summarize the protection afforded by estrogen as well as discuss several possible mechanisms of protection for exogenous estrogen administration.

Estrogen Protects the Heart from Ischemia-reperfusion Injury Via COX-2-derived PGI2

Journal of Cardiovascular Pharmacology. Sep, 2008  |  Pubmed ID: 18806603

There is an accumulating body of data to suggest that estrogen mediates its cardioprotective effects via cyclooxygenase activation and synthesis of prostaglandins (PG), specifically PGI2. We hypothesized that inhibition of COX-2 would prevent estrogen's cardioprotective effects after myocardial ischemia-reperfusion. Acute treatment with 17beta-estradiol (E2; 20 microg/rabbit) increased COX-2 protein expression and activity in the myocardium. To determine the effects of COX-2 inhibition on infarct size after E2 treatment, New Zealand white rabbits were anesthetized and administered the COX-2 inhibitor nimesulide (5 mg/kg) or vehicle intravenously 30 minutes before an intravenous injection of E2. Thirty minutes after estrogen treatment, the coronary artery was occluded for 30 minutes followed by 4 hours of reperfusion. E2 significantly decreased infarct size as a percent of area at risk when compared to vehicle (18.9 +/- 3.1 versus 47.0 +/- 4.1; P < 0.001). Pretreatment with nimesulide nullified the infarct size sparing effect of E2 (55.8 +/- 5.6). Treatment with the PGI2 receptor antagonist RO3244794 also abolished the protective effects of E2 (45.3 +/- 4.5). The results indicate that estrogen protects the myocardium from ischemia-reperfusion injury through increased production of COX-2-derived PGI2. The data indicate that selective COX-2 inhibitors might counteract the potential cytoprotective effects of estrogen in premenopausal or postmenopausal women.

Cardiac Arrhythmia and Injury Induced in Rats by Burst and Pulsed Mode Ultrasound with a Gas Body Contrast Agent

Journal of Ultrasound in Medicine : Official Journal of the American Institute of Ultrasound in Medicine. Nov, 2009  |  Pubmed ID: 19854967

Premature complexes (PCs) in the electrocardiogram (ECG) signal have been reported for myocardial contrast echocardiography and also for burst mode (physical therapy) ultrasound with gas body contrast agents at lower peak rarefactional pressure amplitudes (PRPAs). For contrast echocardiography, irreversibly injured cardiomyocytes have been associated with the arrhythmia. The objective was to determine whether cardiomyocyte injury is associated with the PCs induced by the burst mode at lower PRPAs.

Coxibs Interfere with the Action of Aspirin by Binding Tightly to One Monomer of Cyclooxygenase-1

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2010  |  Pubmed ID: 19955429

Pain associated with inflammation involves prostaglandins synthesized from arachidonic acid (AA) through cyclooxygenase-2 (COX-2) pathways while thromboxane A(2) formed by platelets from AA via cyclooxygenase-1 (COX-1) mediates thrombosis. COX-1 and COX-2 are both targets of nonselective nonsteroidal antiinflammatory drugs (nsNSAIDs) including aspirin whereas COX-2 activity is preferentially blocked by COX-2 inhibitors called coxibs. COXs are homodimers composed of identical subunits, but we have shown that only one subunit is active at a time during catalysis; moreover, many nsNSAIDS bind to a single subunit of a COX dimer to inhibit the COX activity of the entire dimer. Here, we report the surprising observation that celecoxib and other coxibs bind tightly to a subunit of COX-1. Although celecoxib binding to one monomer of COX-1 does not affect the normal catalytic processing of AA by the second, partner subunit, celecoxib does interfere with the inhibition of COX-1 by aspirin in vitro. X-ray crystallographic results obtained with a celecoxib/COX-1 complex show how celecoxib can bind to one of the two available COX sites of the COX-1 dimer. Finally, we find that administration of celecoxib to dogs interferes with the ability of a low dose of aspirin to inhibit AA-induced ex vivo platelet aggregation. COX-2 inhibitors such as celecoxib are widely used for pain relief. Because coxibs exhibit cardiovascular side effects, they are often prescribed in combination with low-dose aspirin to prevent thrombosis. Our studies predict that the cardioprotective effect of low-dose aspirin on COX-1 may be blunted when taken with coxibs.

Thrombogenesis with Continuous Blood Flow in the Inferior Vena Cava. A Novel Mouse Model

Thrombosis and Haemostasis. Aug, 2010  |  Pubmed ID: 20589322

Several rodent models have been used to study deep venous thrombosis (DVT). However, a model that generates consistent venous thrombi in the presence of continuous blood flow, to evaluate therapeutic agents for DVT, is not available. Mice used in the present study were wild-type C57BL/6 (WT), plasminogen activator inhibitor-1 (PAI-1) knock out (KO) and Delta Cytoplasmic Tail (DCT). An electrolytic inferior vena cava (IVC) model (EIM) was used. A 25G stainless-steel needle, attached to a silver coated copper wire electrode (anode), was inserted into the exposed caudal IVC. Another electrode (cathode) was placed subcutaneously. A current of 250 muAmps over 15 minutes was applied. Ultrasound imaging was used to demonstrate the presence of IVC blood flow. Analyses included measurement of plasma soluble P-selectin (sP-Sel), thrombus weight (TW), vein wall morphometrics, P-selectin and Von Willebrand factor (vWF) staining, transmission electron microscopy (TEM), scanning electron microscopy (SEM); and the effect of enoxaparin on TW was evaluated. A current of 250 muAmps over 15 minutes consistently promoted thrombus formation in the IVC. Plasma sP-Sel was decreased in PAI-1 KO and increased in DCT vs. WT (WT/PAI-1: p=0.003, WT/DCT: p=0.0002). Endothelial activation was demonstrated by SEM, TEM, P-selectin and vWF immunohistochemistry and confirmed by inflammatory cell counts. Ultrasound imaging demonstrated thrombus formation in the presence of blood flow. Enoxaparin significantly reduced the thrombus size by 61% in this model. This EIM closely mimics clinical venous disease and can be used to study endothelial cell activation, leukocyte migration, thrombogenesis and therapeutic applications in the presence of blood flow.

Are ECG Premature Complexes Induced by Ultrasonic Cavitation Electrophysiological Responses to Irreversible Cardiomyocyte Injury?

Ultrasound in Medicine & Biology. Feb, 2011  |  Pubmed ID: 21257092

The objective of this study was to explore the relationship between premature complexes (PCs) in the electrocardiogram (ECG) and lethal injury of cardiomyocytes induced by ultrasound exposure of the heart with contrast-agent gas bodies in the circulation. Anesthetized rats were exposed in a heated water bath to 1.55 MHz focused ultrasound with bursts triggered at end systole during contrast agent infusion. PCs were detected in ECG recordings and cardiomyocyte necrosis was scored by identifying Evans blue-stained cells in multiple frozen sections. With 0.1 μL/kg/min infusion of contrast agent for 5 min, both effects increased strongly for 2-ms bursts with increasing peak rarefactional pressure amplitude >1 MPa. At 8 MPa, statistically significant effects were found even for no agent infusion relative to sham tests. For 2-ms bursts at 2 MPa, the highly significant bioeffects seen for 10-, 1- and 0.1-μL/kg/min infusion became marginally significant for 0.01 μL/kg/min, which indicated a lower probability of cavitation nucleation. Burst duration variation from 0.2-20 ms produced no substantial trends in the results. Overall, the two effects were well correlated (r(2) = 0.88). The PCs occurring during contrast-enhanced ultrasound therefore appear to be electrophysiological responses to irreversible cardiomyocyte injury induced by ultrasonic cavitation.

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