Regulation of NF-κB activity by competition between RelA acetylation and ubiquitination.

The nuclear factor (NF)-κB transcription factor has essential roles in inflammation and oncogenesis. Its ubiquitous RelA subunit is regulated by several post-translational modifications, including phosphorylation, ubiquitination and acetylation. Ubiquitination promotes the termination of RelA-dependent transcription, but its regulation is incompletely understood. Through mass spectrometry analysis of ubiquitinated RelA, we identified seven lysines that were attached to degradative and non-degradative forms of polyubiquitin. Interestingly, lysines targeted for acetylation were among the residues identified as ubiquitin acceptor sites. Mutation of these particular sites resulted in decreased polyubiquitination. Acetylation and ubiquitination were found to inhibit each other, consistent with their use of overlapping sites. Reconstitution of rela(-/-) fibroblasts with wild-type and mutant forms of RelA revealed that modifications at these residues can have activating and inhibitory functions depending on the target gene context. Altogether, this study elucidates that ubiquitination and acetylation can modulate each other and regulate nuclear NF-κB function in a gene-specific manner.

The acute effect of IABP-induced pulsatility on coronary vascular resistance and graft flow in critical ill patients during ECMO.

AIM: The combination of the two cardiac support mechanisms of intra-aortic balloon pumping (IABP) and non-pulsatile circulatory extracorporeal membrane oxygenation (ECMO) has been confirmed to improve efficacy of the cardiac support as a whole. However, reports on benefits of diastolic augmentation on coronary vascular bed and graft flowmetry during concomitant use of IABP and ECMO are lacking. The aim of this study was to evaluate the acute impact of IABP support on coronary vascular resistance (CVR) and coronary bypass flows (CBF) in high-risk patients with peripheral ECMO following coronary artery bypass grafting (CABG). METHODS: In eight emergency CABG patients (mean age=67.8±1.9 years; gender: six male and two female; EF=25.5±2.4%) requiring mechanical circulatory support with ECMO hemodynamic parameters, CVR, CBF, diastolic filling index (DFI), graft flow reserve (GFR), and pulsatility index (PI) were analyzed with and without diastolic augmentation using a transit time flowmeter. RESULTS: The addition of IABP to ECMO decreased CVR significantly by 6.5%±1.9% compared to baseline with ECMO alone (1.62±0.2 versus 1.78±0.2; P<0.0045). Accordingly, significant higher mean CBF were found during IABP assist, resulting in a 21.6%±2.6% increase (60.7±8.7 mL/min with versus 51.3±7.4 mL/min without IABP; P<0.0001). IABP also significantly increased DFI by 9.8±0.9% (73.2%±1.4% with versus 66.7%±1.3% without IABP; P<0.0001). GFR was recruited during IABP in all grafts (GFR>1). There were no statistically significant differences in PI with and without IABP assistance (2.6±0.1 versus 2.5±0.2). CONCLUSION: IABP-induced pulsatility significantly improves diastolic filling index and mean coronary bypass graft flows by lowering coronary vascular resistance during non-pulsatile peripheral ECMO. The combination of ECMO with IABP may provide more optimal myocardial oxygen conditions resulting in an improved efficacy of the cardiac support as a whole in critical ill patients with postcardiotomy myocardial dysfunction following CABG.

Myocardial protection in cardiac surgery patients requiring prolonged aortic cross-clamp times: a single-center evaluation of clinical outcomes comparing two blood cardioplegic strategies.

AIM: The aim of this study was to evaluate the impact of intermittent warm (IWC) versus intermittent cold blood cardioplegia (ICC) in high-risk patients that require prolonged periods of aortic cross-clamping during on-pump cardiac surgery. METHODS: From 3527 consecutive patients undergoing on-pump cardiac surgery, 520 patients were retrospectively identified that required prolonged aortic cross-clamp ≥ 75 min. Myocardial protection was performed with ICC (N.=280) or IWC (N.=240). Groups were compared regarding clinical outcomes, myocardial injury (CK-MB, cTnT) and multivariate analysis was performed to assess the impact of applied cardioplegia on 30-day all-cause mortality, cardiac death, perioperative myocardial injury (PM) and major adverse cardiac events (MACE). RESULTS: Demographic data, mean logistic Euroscore, aortic-cross-clamping and CPB time were comparable between groups. Patients with ICC needed more intraoperative defibrillations, had more postoperative blood transfusions and a prolonged hospital stay when compared to the IWC-group (P < 0.05). Thirty-day all-cause mortality tended to be higher in IWC (11% vs. 6%; P = 0.083) with significantly higher cardiac mortality (9% vs. 4%; P=0.015) compared to ICC. Myocardial injury was more pronounced in the IWC-group with a higher incidence of PMI (IWC: 17% vs. ICC:6%; P < 0.05) and MACE (IWC:37% vs. ICC:25%; P < 0.05). Groups did not differ regarding other postoperative clinical outcomes. Multivariate analysis revealed IWC to be independently predictive (P < 0.05) for 30-day all-cause mortality (OR:2.42; 95% CI:1.04-5.05), cardiac death (OR:3.57; 95% CI:1.49-8.85), MACE (OR:1.87; 95% CI:1.22-2.87) and PMI (OR:3.46; 95% CI:1.86-6.41). CONCLUSION: ICC results in less myocardial damage and reduced postoperative cardiac mortality and morbidity in patients requiring extended periods of aortic-cross-clamping during on-pump cardiac surgery, suggesting superior cardioprotection when compared to IWC.

Partial mechanical long-term support with the CircuLite Synergy pump as bridge-to-transplant in congestive heart failure.

BACKGROUND: Full mechanical support with a left ventricular assist device (LVAD) is often limited to very sick patients, as the only survival option. This European multicenter study analyzes the effect of partial mechanical support as bridge-to-transplant in a less sick heart failure patient group. METHODS: The CircuLite Synergy device is implanted via a small right-sided thoracotomy with an inflow cannula in the left atrium and an outflow graft connected to the right subclavian artery without the use of extracorporeal circulation. The pump itself sits in a "pacemaker" pocket subcutaneously in the right clavicular groove. It is able to pump up to 3.0 l/min and partially unload the left ventricle. RESULTS: The device was implanted in 25 patients on the cardiac transplant waiting list (20 males), aged 55.5 +/- 9.6 yrs with an ejection fraction of 21.6 +/- 6.0 %, a mean arterial pressure of 73.5 +/- 8.5 mmHg, a pulmonary capillary wedge pressure of 27.2 +/- 7.8 mmHg and cardiac index of 1.9 +/- 0.4 l/min/m (2). Duration of support ranged from 6 to 238 days. Right heart catheterization showed significant hemodynamic improvement in the short- and intermediate-term after implantation with increases in arterial pressure from 72.6 +/- 11.0 to 79.4 +/- 8.6 mmHg ( P = 0.04) and in cardiac index from 2.0 +/- 0.4 to 2.7 +/- 0.6 l/min/m (2) ( P = 0.003) with a reduction in pulmonary capillary wedge pressure from 28.5 +/- 6.0 to 19.7 +/- 6.9 mmHg ( P = 0.012). CONCLUSIONS: The CircuLite Synergy device is a partial support pump, which is easy to implant and which provides hemodynamic benefits in bridging heart failure patients to cardiac transplant.

Inhibition of the alpha1beta1 isoform of the Na, K-ATPase by 8-methoxycoumestrol without positive inotropic effect in human myocardium--novel aspects of cardiac glycoside pharmacology.

BACKGROUND: The Na,K-ATPase (NKA) is necessary for maintaining the resting membrane potential by transporting Na and K ions across the cell membrane. Although its 3 isoforms expressed in human heart (alpha1beta1, alpha2beta1, and alpha3beta1) possess similar biochemical properties, their specific functions in human tissues remain unknown. In our search for an isoform-specific agent, which can serve to identify isoform-specific functions, we examined 8-methoxycoumestrol in its ability to inhibit the NKA and to produce inotropism in connection with the possibility to identify the NKA isoform-specific functions. METHODS AND RESULTS: In radioligand binding experiments (membrane preparations of yeast expressing isoforms alpha1beta1, alpha2beta1, and alpha3beta1; backdoor phosphorylation; and [H]-ouabain, n = 3), 8-methoxycoumestrol (1-10 microM) produced no or only little inhibition of specific ouabain binding. However, when NKA activity of the alpha1beta1 isoform was measured in membrane preparations from human kidney (reduced form of nicotinamide adenine dinucleotide-coupled assay, n = 3), a concentration-dependent full inhibition of the activity was induced by 8-methoxycoumestrol (IC50: 90 +/- 97 nM), similar to that observed for classical cardiac glycosides digitoxin, digoxin, methyldigoxin, and beta-acetyldigoxin (IC50 = 287 +/- 190 nM, 409 +/- 171 nM, 282 +/- 482 nM, 587 +/- 135 nM, P > 0.05). However, unlike the classical cardiac glycosides, 8-methoxycoumestrol did not increase cardiac contractility of electrically stimulated human right atrial trabeculae. CONCLUSIONS: These results indicate that 8-methoxycoumestrol inhibits the human alpha1beta1 NKA by a mechanism different to that of cardiac glycosides. In addition, the inhibition of the alpha1beta1 NKA activity seems not sufficient to evoke positive inotropy in human trabeculae, indicating that either the positive inotropic effect of cardiac glycosides is not mediated via the alpha1beta1 isoform or the specific glycoside binding to alpha1beta1 is needed for positive inotropy.

Pre-ischaemic exogenous surfactant reduces pulmonary injury in rat ischaemia/reperfusion.

The optimal timing of exogenous surfactant application to reduce pulmonary injury and dysfunction was investigated in a rat lung ischaemia and reperfusion injury model. Lungs were subjected to flush perfusion, surfactant instillation, cold ischaemia (4 degrees C, 4 h) and reperfusion (60 min). Animals received surfactant before (group 1) or at the end (2) of ischaemia, or during reperfusion (3) or not at all (4). Control groups included "worst case" without Perfadex and surfactant (5), "no injury" without (6) or with surfactant (7), and ischaemia with pre-ischaemic surfactant (8). Intra-alveolar oedema and blood-air barrier injury were estimated by light and electron microscopic stereology. Perfusate oxygenation and pulmonary arterial pressure (P(pa)) were determined during reperfusion in groups 1 to 4. Intra-alveolar oedema was almost absent in groups 1, 6, 7 and 8, pronounced in 2, 3 and 4, and severe in 5. Blood-air barrier injury was moderate in groups 1 and 8, slightly pronounced in 2, 3 and 4, extensive in 5 and almost absent in 6 and 7. Perfusate oxygenation was significantly higher in group 1 compared with groups 2 to 4. P(pa) did not differ between the groups. In conclusion, exogenous surfactant attenuates intra-alveolar oedema formation and blood-air barrier damage and improves perfusate oxygenation in the rat lung, especially when applied before ischaemic storage.

Preischemic iloprost application for improvement of graft preservation: which route is superior in experimental pig lung transplantation: inhaled or intravenous?

BACKGROUND: Optimal allograft protection is essential in lung transplantation to reduce postoperative organ dysfunction. Although intravenous prostanoids are routinely used to ameliorate reperfusion injury, the latest evidence suggests a similar efficacy of inhaled prostacyclin. Therefore, we compared donor lung-pretreatment using inhaled lioprost (Ventavis) with the commonly used intravenous technique. METHODS: Five pig lungs were each preserved with Perfadex and stored for 27 hours without (group 1) or with (group-2, 100 prior aerosolized of iloprost were (group 3) or iloprost (IV). Following left lung transplantation, hemodynamics, Po(2)/F(i)o(2), compliance, and wet-to-dry ratio were monitored for 6 hours and compared to sham controls using ANOVA analysis with repeated measures. RESULTS: The mortality was 100% in group 3. All other animals survived (P < .001). Dynamic compliance and PVR were superior in the endobronchially pretreated iloprost group as compared with untreated organs (P < .05), whereas oxygenation was comparable overall W/D-ratio revealed significantly lower lung water in group 2 (P = .027) compared with group 3. CONCLUSION: Preischemic alveolar deposition of iloprost is superior to IV pretreatment as reflected by significantly improved allograft function. This strategy offers technique to optimize pulmonary preservation.

Selective cerebral perfusion via right axillary artery direct cannulation for aortic arch surgery.

BACKGROUND: The risk of neurological complications is still a life-threatening event for patients undergoing proximal aortic arch or total aortic arch surgery. To prevent these complications, axillary artery cannulation and antegrade selective cerebral perfusion were utilized. We compared the effects of using hypothermic circulatory arrest (HCA) alone or with selective cerebral perfusion (SCP/AX) via right side axillary artery direct cannulation. METHODS: 120 patients, mean age 61 +/- 12 years (range 26 - 80), underwent proximal aortic or total aortic arch replacement between 1999 and 2004; 46 were female. We retrospectively compared the results of the two patient groups comparable for preoperative risk factors: 71 pts were operated using HCA beginning in 1999 and 49 pts using HCA/SCP via axillary artery direct cannulation since 2002. The indication for surgery was an aortic aneurysm in 80 (67 %) patients and aortic dissection in 36 (30 %) patients. The groups were well matched with regard to median age (60 vs. 62 yrs), urgency (emergent/urgent 36 vs. 44 %; elective 64 vs. 65 %), and several other known risk factors ( p = ns). RESULTS: Overall in-hospital mortality was 13 %: 10 % with HCA vs. 6 % with SCP/AX. Permanent neurological dysfunction occurred in 10 % with HCA vs. 6 % with SCP/AX. Transient neurological dysfunction (TND) in patients surviving without stroke was lower with SCP/AX (10 %) than with HCA (17 %) ( p = ns). Mean duration of HCA was 28 +/- 12 min when isolated HCA was used, and significantly shorter with 21 +/- 6 min when the combination of SCP/AX ( p = 0.03) was used. Mean duration of CPB was 202 +/- 55 min with HCA vs. 192 +/- 50 min with SCP/AX ( p = ns). Comparison of the groups who had comparable preoperative risk factors showed a trend towards lower in-hospital mortality, stroke and TND rates, a significant reduction in cardiac ( p = 0.034), infectious ( p = 0.025) and bleeding complications ( p = 0.04) in SCP/AX compared with HCA, as well as a significantly shorter duration of hospitalization ( p = 0.046) and shorter ICU stay ( p = ns). CONCLUSION: Our results suggest that HCA/SCP is superior to HCA alone for preventing cerebral injury during operations on the aortic arch. By reducing embolic risk, as well as the duration of HCA, SCP with axillary artery direct cannulation may be the optimal technique for averting cerebral events, reducing complications, and shortening hospital stays following aortic arch repair.

Are alterations of lymphocyte subpopulations in polymicrobial sepsis and DHEA treatment mediated by the tumour necrosis factor (TNF)-alpha receptor (TNF-RI)? A study in TNF-RI (TNF-RI(-/-)) knock-out rodents.

Sepsis is associated with depression of T cell-dependent immune reactivity with proinflammatory cytokines, such as tumour necrosis factor (TNF)-alpha, playing an important role. Recent investigations describe an association between these immunological alterations and disturbances of the endocrine system, related most frequently to sex steroid hormones. Dehydroepiandrosterone (DHEA), one of the most abundant adrenal sex steroid precursors, seems to have a protective immunological effect towards septic insults. In this study, both the role of TNF-receptor I (RI) and possible interactions in the protective role of DHEA were investigated in a murine model of polymicrobial sepsis. Polymicrobial sepsis was induced by caecal ligation and puncture (CLP) in a murine model. The effects of DHEA on survival, clinical parameters and cellular immunity (T lymphocytes and natural killer (NK) cells) were investigated. CLP was performed in genetically modified TNF-RI knock-out (TNF-RI(-/-)) and genetically unmodified (wild-type, WT) mice. DHEA application was associated with a decrease in the mortality rate in WT animals. A mortality rate of 91.7% was observed in TNF-RI(-/-) mice after CLP. This mortality rate was reduced to 37.5% by the application of DHEA. In sham-operated TNF-RI(-/-) animals, a significantly higher proportion of NK cells within the lymphocyte population was measured compared with the corresponding WT group. After CLP, a significant increase in the percentage cell count of NK cells was recorded in WT mice. Overall, following DHEA application in WT mice, an alteration in the cellular immune response was characterized by a reduction in the percentage counts of CD4(+), CD8(+) and NK cells. In the group of TNF-RI(-/-) mice treated with DHEA, no increase in the percentage cell count of NK cells was observed after CLP. No data for cell analysis were available from the CLP-TNF-RI(-/-) mice treated with saline, due to the high mortality rate in these animals. DHEA reduces the complications of sepsis in a TNF-RI-independent manner. Our study suggests that NK cells are involved in the protective mechanism of DHEA in WT mice. It would therefore seem that DHEA represents a feasible alternative therapy for the dysregulated immune system in sepsis.

Evaluation of pulmonary edema: stereological versus gravimetrical analysis.

Assessment of lung edema by gravimetrical analysis is a standard method to evaluate the severity of experimentally induced ischemia/reperfusion (IR) injury. The aim of this study was to compare gravimetrical assessment of pulmonary edema with a stereological approach which allows for qualitative and quantitative distinction between intravascular and edematous fluids by light microscopy. Eight experimental groups which differed in mode of preservation, ischemic storage and pharmacological treatments were studied in an extracorporeal rat lung model. Analysis of the pooled data showed that the wet/dry ratio values mainly reflected the amount of intra-alveolar edema (r(s) = 0.442; p = 0.0057) but only stereological assessment of edema formation revealed differences depending on the treatment used. Only stereological data correlated significantly with oxygen tension measured at the end of reperfusion (r(s) = -0.530; p = 0.0009). We conclude that gravimetry is of minor functional importance compared to assessment by stereological methods which prove to be a reliable and efficient tool for the evaluation of IR injury in the different experimental settings.

Renal carcinoma with caval vein infiltration and triple coronary disease: one-stage surgical management.

We report the one-stage surgical management in a 68-year-old patient with a renal cell carcinoma with extended intravascular growth into the inferior vena cava combined with severe triple coronary artery disease. After nephrectomy the resection of the intravascular tumor and caval reconstruction were performed in deep hypothermic circulatory arrest. Coronary revascularization was accomplished while rewarming. The postoperative course was uneventful. Nine months after this operation there are no signs of reoccurrence.

Nitroglycerin alters alveolar type II cell ultrastructure after ischemia and reperfusion.

BACKGROUND: Although administration of nitric oxide (NO) has been suggested to reduce pulmonary reimplantation response, concerns remain about cytotoxic side effects. METHODS: Using light and electron microscopy, we examined the effects of the NO donor nitroglycerin (NTG) (0.1 mg/ml) as a supplement to the preservation solution Celsior on the structural integrity of rat lungs after extracorporeal ischemia (4 hours at 10 degrees C) and reperfusion (50 minutes) (IR). We performed evaluation in comparison with Celsior alone after IR using either standard antegrade perfusion through the pulmonary artery or retrograde perfusion through the left atrium as an alternative way to improve the preservation quality. Untreated, non-ischemic lungs served as controls (n = 5 per group). We recorded respiratory and hemodynamic parameters during reperfusion. Tissue collection using systematic uniform random sampling was representative for the whole organ and allowed stereologic quantification of structures. RESULTS: After IR, histochemistry revealed no breaks in the alveolo-capillary barrier and we detected no alveolar flooding. Edema formed in the peribronchovascular cuffs, of which the volume fraction was increased (p =.008). Vasoconstriction of the smaller arteries accompanied antegrade flush, which occurred neither after administration of NTG nor after retrograde flush, as shown by immunostaining for alpha-smooth muscle actin. Treatment with NTG was associated with focal disintegration of Type II cells, which displayed edematous swelling of distinct cell compartments and lysis of mitochondria and cells. Nitroglycerin prevented alveolar collapse, which was increased in the other IR groups (p = 0.013). We observed alterations in intra-alveolar surfactant components. CONCLUSION: These findings indicate pathologic effects of NTG treatment on alveolar epithelial integrity. Therefore, we suggest further critical evaluation of NTG/NO for therapeutic use in lung transplantation.

Aerosolized iloprost for severe pulmonary hypertension as a bridge to heart transplantation.

Preexisting pulmonary hypertension in pediatric patients is associated with poor outcome after cardiac transplantation because of donor right ventricular dysfunction. To avoid a combined heart-lung transplantation in a 17-year-old patient, we used an intensified pretreatment with intravenous prostacyclin and dobutamine combined with an inhalative therapy with the aerosolized prostacyclin-analog Iloprost. With this regimen, the patient was hemodynamically stabilized for the waiting period of 21 days after which an uneventful cardiac transplantation was performed.

Impact of PAF antagonist BN 52021 (Ginkolide B) on post-ischemic graft function in clinical lung transplantation.

BACKGROUND: Platelet activating factor (PAF) is associated with ischemia/reperfusion injury (I/R) after lung transplantation. Following promising experimental results, this prospective trial investigated the potential effect of PAF antagonist BN 52021 (ginkolide B) on clinical Euro-Collins (EC)-based lung preservation. METHODS: We analyzed 8 double-lung transplant patients in each of 3 groups. In the low-dose group (LDG), donor lungs were perfused with EC containing 2 mg/kg BN 52021, whereas we used 10 mg/kg in the high-dose group (HDG) and placebo in the control group (CG). Before reperfusing the first lung, we administered intravenously 120 mg BN 52021 (LDG), 600 mg BN 52021 (HDG), or placebo (CG). Hemodynamics in terms of pulmonary arterial pressure, pulmonary vascular resistance and serial determinations of the alveolo-arterial oxygen difference (AaDO(2)) were recorded. We measured blood levels of PAF pre-operatively and post-operatively, after 10 minutes and after 3, 8, 24, 48, and 144 hours. RESULTS: Within 32 hours, we noted a tendency toward better AaDO(2) in the LDG and the HDG compared with the CG (p > 0.05). We observed a significant improvement of AaDO(2) after 3 hours (HDG, p = 0.033) and 8 hours (LDG, p = 0.024), with poorest values in the CG. The PAF concentrations were lowest in the HDG, with significant deterioration 10 minutes after reperfusion. In contrast, placebo led to higher PAF levels. We measured significantly lower PAF concentrations (HDG vs CG) at 10 minutes and at 6 days post-operatively. CONCLUSIONS: Use of high-dose PAF antagonist BN 52021 can easily be combined with clinical preservation methods and may help optimize pulmonary function with reduced PAF levels, in the early post-ischemic period.

Ten years experience with lung and heart-lung transplantation in primary and secondary pulmonary hypertension.

OBJECTIVE: Patients with primary pulmonary hypertension (PPHT) have a worse natural outcome compared with those with secondary pulmonary hypertension in Eisenmenger's syndrome (ES) and chronic pulmonary embolism (CPE). Lung transplantation (SLTx, DLTx, HLTx) still remains the only therapeutical option for patients with this type of endstage lung disease. METHODS: From 1988 to 1998, 63 patients underwent lung transplantation for PPHT (n=29, 9 m, 20 f, 2 SLTx, 14 DLTx, 13 HLTx), ES (n=29, 13 m, 16 f, 2 SLTx, 3 DLTx, 24 HLTx) or CPE (n=5, 2 m, 3 f, 1 SLTx, 2 DLTx, 2 HLTx). Groups were comparable for NYHA functional class, preoperative pulmonary arterial pressure, recipient and donor age, ischemic time, necessity and duration of cardiopulmonary bypass and cross-match. RESULTS: The 1-, 3- and 5-year survival was 52, 40 and 35% for the PPHT-group, 83, 78 and 74% for the ES-group and 80, 60 and 60% for the CPE-group, respectively (P=0.026, P=0.033, P=0.082 for 1-, 3- and 5-year survival). Patients following DLTx showed a lower 1-year survival rate as compared with patients after HLTx both in PPHT patients (36 vs. 62%, P=0.091) and in ES patients (67 vs. 83%, P=0.213). The incidence of bronchiolitis obliterans syndrome was 29% at 1 year and 45% at 3 years for the PPHT-group vs. 17 and 65% for the ES-group (n. s. in between groups). Excluding postoperative ventilation time (PPHT-group: 26.8+/-24.0 days vs. ES-group: 16.1+/-30.8 days, P=0. 011) and a higher incidence of infectious causes of death (PPHT-group n=8 vs. ES-group n=1, P=0.017) groups were comparable with regard to their postoperative courses. CONCLUSIONS: It is concluded, that predominantly the underlying primary disease influences graft survival after lung transplantation in patients with pulmonary hypertension compared with all other patient and procedure dependent factors. Lung transplantation in patients with PPHT requires further investigations to achieve results comparable with other indications.