Nicole Conrad

Inducible nitric oxide synthase activation after ischemia/reperfusion contributes to myocardial dysfunction and extent of infarct size in rabbits: evidence for a late phase of nitric oxide-mediated reperfusion injury

BACKGROUND Ischemia/reperfusion (I/R) leads to the induction of inducible nitric oxide synthase. The present study investigated the effects of selective and continuous inhibition of iNOS on myocardial performance, infarct size and histomorphological changes after I/R in rabbits. METHODS AND RESULTS Ischemia/reperfusion (I/R) was induced by occlusion of the circumflex coronary artery for 30 min followed by 48 h of reperfusion. Sham animals (group A) served as control. Three groups were subjected to I/R: (B) placebo; (C) aminoguanidine (AMG; 10 mg/kg bolus) given prior to and 48 h after I/R to test its acute effects; (D) AMG (300 mg/kg/day s.c.) to test effects of continuous treatment. Hemodynamics, myocardial blood flow, infarct size, iNOS activity, cGMP levels, immunohistochemical analysis of iNOS expression and AMG tissue levels were determined. Continuous AMG treatment improved myocardial performance (hemodynamics and blood flow) compared to placebo group. iNOS was highest in placebo-treated animals. AMG tissue levels were highest in tissues affected by I/R. Infarct size (% of the circumflex region) was significantly smaller in group D when compared to group B. CONCLUSIONS This is the first study showing that activation of myocardial iNOS isozyme during 48 h of reperfusion contributes to a late phase of I/R-induced injury in rabbits. Selective and continuous modulation of iNOS by AMG over this time period exerts protective effects with respect to myocardial performance, coronary blood flow, cellular infiltration and reduction of infarct size; this may be a novel therapeutic approach in the clinical situation to limit irreversible myocardial injury associated with ischemia and reperfusion.

Nitric oxide and endothelin in the development of cardiac allograft vasculopathy. Potential targets for therapeutic interventions

Extensive research has been carried out in recent years to discover the potential risk factors contributing to cardiac allograft atherogenesis. Injury to endothelial cells has been regarded as an important early mechanism in the development of transplant atherosclerosis; it leads to the manifestation of epicardial and microvascular endothelial dysfunction and development of intimal hyperplasia. Moreover, continuous minor endothelial cell damage contributes to endothelial dysfunction which reflects one of the first measurable steps in the cascade of atherogenesis without macroscopic evidence of vascular lesions. The discovery of two important vasoactive substances nitric oxide (NO) and endothelin (ET) has brought new insights but also new unsolved questions regarding the mechanisms leading to atherosclerosis. To date it is known that both substances play a major role in both prevention and development of atherosclerosis. NO appears to be protective in low concentrations by inhibiting leukocyte and platelet activation/adherence and smooth muscle cell proliferation. Impaired endothelial NO production, as one cause of endothelial dysfunction may occur in early stages of atherosclerosis before macroscopic lesions are evident. In addition, increased endothelin release also results in endothelial dysfunction by inducing vasoconstriction; it promotes vascular lesion formation due to endothelial- and vascular smooth muscle cell proliferation. Direct and indirect manipulation of both the NO and ET signal transduction systems may provide novel preventive and therapeutic approaches for limiting transplant atherogenesis and to treat native atherosclerosis. This review summarizes important experimental and clinical evidence which points to nitric oxide and endothelin as potential therapeutic targets in the process of cardiac allograft vasculopathy.

Effects of Celsior and University of Wisconsin preservation solutions on hemodynamics and endothelial function after cardiac transplantation in humans: a single-center, prospective, randomized trial.

Abstract Optimal preservation of the myocardium remains a major concern in clinical and experimental heart transplantation. The present study compared the efficacy of University of Wisconsin (UW) and Celsior preservation solution with respect to myocardial performance, epicardial and microvascular endothelial vasomotor function and myocardial expression of endothelin and nitric oxide synthases in humans. Forty‐one cardiac transplant recipients received either UW (n = 20) or Celsior (n = 21) preserved hearts. Catecholamine and vasodilator requirements were assessed within the first 5 postoperative days. Left ventricular performance and endothelial function was assessed 1 month after transplantation. Endothelin and nitric oxide synthase gene expression were detected in myocardial biopsy samples. Celsior preserved hearts required significantly more catecholamines and vasodilators within the first 5 postoperative days. Myocardial performance and endothelial function were comparable 1 month after transplantation. Total ischemic time correlated with impaired endothelial function in the Celsior but not in the UW group. Endothelin and inducible nitric oxide synthase gene expression were significantly higher in the Celsior group. The results of the study show that both solutions provide myocardial protection with regard to left ventricular performance and endothelial function 1 month after cardiac transplantation. The necessity for higher vasodilator and catecholamine therapy in Celsior preserved hearts suggests post‐ischemic myocardial stunning within the first 5 postoperative days. The positive correlation between impaired endothelial function and total ischemic time in the Celsior group requires longitudinal investigation in particular with regard to the development of allograft vasculopathy.

An association between microvascular endothelial dysfunction, transcardiac nitric oxide production and pro-inflammatory cytokines after heart transplantation in humans

Abstract Endothelial dysfunction anticipates the development of transplant coronary artery disease (TxCAD) observed more than 1 year after transplantation (HTx). We investigated whether in patients early after HTx myocardial inducible and constitutive nitric oxide synthases (iNOS; cNOS) are expressed and cardiac nitric oxide production occurs. Moreover, a possible relationship to alterations in endothelium dependent and independent vasomotor function was assessed. Forty‐two transplant recipients were studied 37 ± 5 days after HTx. Microvascular coronary flow velocity reserve (CFVR) was tested endothelium dependent (acetylcholine; 30 μg/min × 5 min/i.c.) and independent (adenosine; 160 μg/min × 5 min/i.c.) by Doppler flow wire. Flow velocity increase by a factor greater than 2 was considered normal. Quantitative coronary angiography was used to assess epicardial vasomotor function in response to the same stimuli. Myocardial iNOS and cNOS gene expression were detected by semiquantitative reversed transcriptase polymerase chain reaction. Plasma nitrite levels (μM) were measured by spectrophotometry. Cytokines (TNF‐a, IL‐6; pg/ml) were measured by enzyme linked immunosorbent assay. In 26.1% of patients (n = 11; group A) an impaired endothelium dependent CFVR (1.65 ± 0.23 increase) was observed; in 73.9 % (n = 31, group B) a normal endothelium dependent CFVR (3.0 ± 0.7 increase; P = 0.003) was observed. Myocardial iNOS and cNOS gene expression did not differ between the two groups. Transcardiac cytokine production was noted in 58.8% of patients for IL‐6 and in 53.3% for TNF‐α. Coronary sinus (CS) levels of TNF‐α, IL‐6 and nitrite were higher in group A. A significant increase in nitrite production was found only in patients with impaired endothelium dependent CFVR (aorta: 43.9 ± 3.7 vs CS: 52.8 ± 5.6, P = 0.05), suggesting transcardiac nitric oxide production. In addition, CS nitrite levels correlated with CS TNF‐a levels in patients with impaired CFVR (r = 0.44, P = 0.003). Microvascular endothelium dependent CFVR is impaired in 26% of patients early after HTx. Activation of cytokines and the NO pathway seem to be involved in this vasomotor dysfunction The association between cardiac nitric oxide production and TNF‐a in this group indicates a chronic high immunologic process, which may represent an early and important target for therapy and prevention of TxCAD.

Coronary flow reserve and nitric oxide synthases after cardiac transplantation in humans

OBJECTIVE Coronary endothelial dysfunction may precede morphological changes in both the epicardial conduit and microvascular resistance vessels in heart transplant recipients. Since the development of transplant atherosclerosis is the major limiting factor for long-term survival, the identification of early mediators of vasomotor dysfunction may be of therapeutic interest. We therefore investigated the potential relationship between the expression of nitric oxide synthases (NOS) and coronary endothelial function in human cardiac transplant recipients over time. METHODS Forty-two human cardiac transplant recipients were studied at 1 and 12 months after heart transplantation (HTx). The microvascular coronary flow velocity reserve (CFVR) was tested for endothelium-dependent (acetylcholine) and -independent (adenosine) stimuli by intravascular Doppler flow-wire. Epicardial diameter changes were evaluated by quantitative coronary angiography. Endomyocardial inducible (iNOS) and endothelial constitutive nitric oxide synthase were determined by RT-PCR. Nitric oxide production (nitrite and nitrate (NOx)) and TNF-alpha were measured in plasma samples from the aorta and coronary sinus. RESULTS CFVR was impaired in 26.1% (n=11) of patients at 1 month and in 31% (n=13) 12 months after HTx. iNOS-mRNA levels were significantly higher in patients with impaired endothelium-dependent CFVR. In addition, only in these patients were TNF-alpha levels higher and these correlated with plasma NOx levels at 1 and 12 months post-HTx (1 month: r=0.81, P=0.001; 12 months: r=0.62, P=0.04). CONCLUSIONS Coronary microcirculatory dysfunction in response to acetylcholine is present in nearly 30% of patients during the first year following transplantation. These patients present with higher iNOS-mRNA expression and TNF-alpha plasma levels. Selective modulation of the TNF-alpha/iNOS-pathway may be of therapeutic value to improve coronary endothelial dysfunction in cardiac transplant recipients.

Expression von TNF-α und löslichen Adhäsionsmolekülen nach koronarchirurgischen Eingriffen mit und ohne extrakorporaler Zirkulation

ZusammenfassungHintergrund Das operative Trauma und die Verwendung der extrakorporalen Zirkulation (EKZ) während herzchirurgischer Eingriffe sind mit einer systemischen inflammatorischen Reaktion des Organismus verbunden. Die vorliegende Studie untersucht die Effekte der EKZ auf die Expression pro-inflammatorischer Zytokine und Adhäsionsmoleküle im Vergleich zu koronarchirurgischen Eingriffen ohne Herz-Lungen-Maschine (OPCAB). Methoden 26 elektive Patienten mit einer koronaren Herzerkrankung (KHK) wurden randomisiert entweder mit Anwendung der EKZ, Hypothermie und Blutkardioplegie oder minimal invasiv am schlagenden Herzen ohne EKZ operiert. Bei allen Patienten erfolgte der Zugang über eine Sternotomie. Die systemischen Spiegel des proinflammatorischen Zytokins TNF-α und der Adhäsionsmoleküle P-Selektin, ICAM-1 (intrazelluläres Adhäsionsmolekül 1) und VCAM-1 wurden gemessen. Ergebnisse Die TNF-α Spiegel waren in der EKZ-Gruppe signifikant höher als in der minimal invasiven Patientengruppe. Sowohl die systemische als auch myokardiale Expression der Adhäsionsmoleküle war in der EKZ-Gruppe deutlich höher. Bei diesen Patienten fand sich ebenfalls eine signifikante Korrelation von TNF-α und Adhäsionsmolekülen. Die Patienten in der EKZ-Gruppe benötigen in den ersten 24 h eine signifikant höhere Katecholaminsubstitution. Schlussfolgerungen Bei vergleichbarem chirurgischen Trauma ist die systemische inflammatorische Reaktion und myokardiale Expression von Adhäsionsmolekülen ohne Anwendung der EKZ deutlich geringer. Dies geht mit einer geringeren Katecholaminsubstitution einher. Da der klinische Verlauf in beiden Patientengruppen vergleichbar ist erscheint die Untersuchung größerer Patientenkollektive sinnvoll um die individuelle Anwendung der jeweiligen Verfahren zu optimieren.SummaryBackground Cardiopulmonary bypass (CPB) and operative trauma are associated with increased expression of proinflammatory mediators. We determined the relative contribution of CPB on activation of cytokines and adhesion molecules in patients undergoing coronary revascularization by comparing them with patients receiving off-pump coronary artery bypass grafting (OPCAB). Methods Twenty-six patients were assigned to either the OPCAB procedure using a suction device and regular sternotomy (n=13) or were treated conventionally using extracorporeal circulation, blood cardioplegia and hypothermic arrest (29–31°C; n=13). Systemic levels of TNF-α and the soluble adhesion molecules P-Selectin, ICAM-1 and VCAM-1 were assayed. Results Systemic expression of adhesion molecules were lower in the OPCAB group. Coronary revascularization with CPB resulted in a significantly higher expression of TNF-α which was associated with P-Selectin and ICAM-1 expression. This was accompanied with higher catecholamine requirement in the CPB group in the early postoperative period. Conclusions Despite comparable surgical trauma, the OPCAB procedure without the use of CPB and cardioplegic arrest significantly reduces systemic and cardiac adhesion molecule expression and catecholamine requirement. Since the clinical course in the early postoperative period was comparable, larger trials are required to select the appropriate patient who benefits most from one or the other treatment regime.

Systemic but not cardiac expression of P-selectin and ICAM-1 mediate coronary microcirculatory changes in cardiac transplant recipients

Purpose: Expression of adhesion molecules mediate endothelial dysfunction which leads to progression of classic atherosclerosis. Endothelial dysfunction is a predictor of transplant vasculopathy (TVP) frequently observed in allografts . 1 year after transplantation (HTx). We investigated the role of P-Selectin and ICAM-1 and alterations of the coronary microvasculature 1 and 12 months (Mo) after HTx. Patients/Methods: Coronary flow reserve (CFR) was measured in response to endothelium dependent (Acetylcholine (Ach); 30 mg/min/5min) and endothelium independent (Adenosine (Ade); 160 mg/min/5 min) stimuli. Expression of adhesion molecules (ng/ml) in aorta and coronary sinus was determined by ELISA 1, 6 and 12 Mo after HTx. In addition, levels of TNF-a (pg/ml), TNF specific receptors (TNF-Rp1 and TNF-Rp2; ng/ml) were determined. Results: Adhesion molecules were highly expressed 1 Mo after HTx and remained at high levels when compared to healthy control subjects (Ko) (ICAM-1, Ko: 140.2612.8; HTP: 1 Mo: 286.7628.3; 6 Mo: 195.8611.3; 12 Mo: 192.4610.2, p,.05 alle HTP vs Ko; P-Selectin: Ko: 34.365.8; HTP: 1 Mo: 78.869.3; 6 Mo: 65.166.8; 12 Mo: 47.362.9; p,.05 all HTx vs Ko). In addition, TNF-a remained high troughout the first yeart (Ko: ; HTx: 1 Mo: 19.562.3; 6 Mo: 21.564.8; 12 Mo: 24.364.1, p,.05 alle HTx vs Ko). A significant inverse correlation was found between CFR and TNF-a (r5.37, p5.03) and TNF-Rp1 (r5.39, p5.02) 12 Mo after HTx. Moreover, a significant correlation was found between TNF-a and P-Selectin (r5 .62, p5.01) 12 Mo after HTx. Conclusions: The association between impaired endothelium dependent CFR, expression/activation of P-Selectin and TNF-a suggests a critical role in the process of chronic rejection and is of potential therapeutic interest in the treatment and prevention of transplant coronary artery disease.