Apolipoprotein E genotype, TNF-α 308G/A and risk for cardiac surgery associated-acute kidney injury in Caucasians.

OBJECTIVES: Acute kidney injury following cardiac surgery depicts a severe clinical problem that is strongly associated with adverse short- and long-term outcome. We analyzed two common genetic polymorphisms that have previously been linked to renal failure and inflammation, and have been supposed to be associated with cardiac surgery associated-acute kidney injury (CSA-AKI). METHODS: A total of 1415 consecutive patients who underwent elective cardiac surgery with CPB at our institution were prospectively enrolled. Patients were genotyped for Apolipoprotein E (ApoE E2,E3,E4) (rs429358 and rs7412) and TNF-α-308 G > A (rs1800629). RESULTS: Demographic characteristics and procedural data revealed no significant differences between genotypes. No association between ApoE (E2,E3,E4) and TNF-α-308 G > A genotypes and the RIFLE criteria could be detected. Several multiple linear regression analyses for postoperative creatinine increase revealed highly significant associations for aortic cross clamp time (p < 0.001), CPB-time (p < 0.001), norepinephrine (p < 0.001), left ventricular function (p = 0.004) and blood transfusion (p < 0.001). No associations were found for ApoE (E2,E3,E4) and TNF-α-308 G > A genotypes or baseline creatinine. When the sample size is 1415, the multiple linear regression test of R(2 )= 0 for seven covariates assuming normal distribution will have at least 99% power with significance level 0.05 to detect an R(2) of 0.108 or 0.107 as observed in the data. CONCLUSIONS: ApoE (E2,E3,E4) polymorphism and the TNF-α-308 G > A polymorphism are not associated with renal injury after CPB.

Tumor necrosis factor-α -863 C/A promoter polymorphism affects the inflammatory response after cardiac surgery.

OBJECTIVE: Cardiac surgery using cardiopulmonary bypass (CPB) initiates an inflammatory response that shows a wide inter-individual range and determines postoperative morbidity. Previous research suggests that genetic diversity contributes to individual susceptibility to perioperative trauma and stress. Nevertheless, the genetic triggering of the tumor necrosis factor-alpha (TNF-α) release remains unclear. We tested two genetic single-nucleotide polymorphisms (SNPs) from the promoter region of the TNF-α gene for associations with perioperative TNF-α level after CPB. METHODS: We prospectively included 122 patients, who underwent elective coronary artery bypass grafting (CABG). Patients were genotyped for TNF-α -863 C/A (rs1800630) and TNF-α -308 G/A (rs1800629). Plasma level of TNF-α was obtained preoperatively, at the end of CPB, 6h postoperatively, and on the first postoperative day (POD). RESULTS: Demographic characteristics and operative data revealed no significant differences between the different genotypes. Multiple linear regression analyses revealed significant associations for the TNF-α 863 C/A polymorphism: the major -863 CC variant was associated with higher TNF-α level preoperatively (p = 0.003), after CPB (p = 0.005), and 6h postoperatively (p = 0.010), independently from CPB time, left ventricle (LV) function and age. Contrarily, the AA allele had lower TNF-α level preoperatively (p = 0.008), after surgery (p = 0.024) and 6h postoperatively (p = 0.001). For the TNF-α 308 G/A polymorphism, only few significant associations could be observed: -308 GG carriers were associated with lower TNF-α level immediately after CPB (p = 0.020), whereas 308 AA carriers were significantly associated with elevated TNF-α level preoperatively (p = 0.032) and immediately after CPB (p = 0.05). No heterozygote variant of both SNPs revealed any significant associations with perioperative TNF-α level. CONCLUSIONS: The current study suggests that the major -863 CC variant determines elevated TNF-α level preoperatively and throughout the postoperative course after CPB.

Identification of an up-regulated anti-apoptotic network in the internal thoracic artery.

BACKGROUND: The radial artery (RA) is known as an atherosclerosis-prone vessel in contrast to the atherosclerosis-resistant internal thoracic artery (ITA). The purpose of the present study was to compare the gene expression profile of these arteries from the same patient in order to identify genes involved in atherogenesis or intimal hyperplasia. METHODS: Paired specimens of RA and ITA (n=6) were analyzed by histomorphometry and whole genome microarray. The microarray data underwent pathway analysis to identify biological networks. Laser microdissection (LMD) was used to identify the cellular expression of candidate genes in the intimal or medial layer of the ITA and RA. RESULTS: Histomorphometric analyses revealed a significantly higher degree of intimal hyperplasia in the RA compared to the ITA. 552 genes were differentially expressed in the ITA and RA. qRT-PCR confirmed a significant up-regulation of six anti-apoptotic genes. p21 (11.8-fold, p=0.011), CCL2 (5.4-fold, p=0.034), SOCS3 (7.2-fold, p=0.002), IER3 (4.1-fold, p=0.048), MCL-1 (2.6-fold, p=0.025) and IL-6 (17.8-fold, p=0.046) were up-regulated in the ITA. LMD confirmed that cells of the intimal layer of the ITA consistently expressed higher levels of all six candidate genes than those of the RA. CONCLUSIONS: Microarray analysis and qRT-PCR identified significantly up-regulated genes in the ITA involved in an anti-apoptotic network. LMD revealed a higher expression of all anti-apoptotic genes in the intimal area of the ITA. These genes may play an important role in protecting the intima of the ITA from developing hyperplasia and atherosclerosis.

Valvular chondromodulin-1 expression is downregulated in a rabbit model of infective endocarditis.

BACKGROUND AND AIM OF THE STUDY: Tissue neovascularization is a major event in the development and dissemination of inflammatory diseases, such as infective endocarditis (IE). Although the molecular triggers which allow vascular ingrowth in the aforementioned avascular regions are not well understood, they may represent potential prophylactic or therapeutic targets. Thus, an investigation was conducted to determine whether the expression of chondromodulin-1 (Chm-1), an anti-angiogenic protein, is dysregulated in mitral valves in a rabbit model of IE, and whether Chm-1 transcripts are differentially expressed in various heart tissues. METHODS: Five groups of animals (seven per group) were studied: group I was untreated controls; group II received (intravenously) 6x10(6) colony-forming units of Staphylococcus aureus; group III underwent mitral valve surgery; and groups IV and V underwent surgery and received S. aureus (as per group II), with tissues sampled at 6 and 12 h after surgery, respectively). Mitral valve surgery was performed by sewing a Dacron patch onto the valve, thereby creating a lesion and causing valve insufficiency. Explanted hearts were dissected and Chm-1 expression was determined using both conventional and real-time RT-PCR. RESULTS: Chm-1 transcripts were found in all cardiac regions, with strong expression in the heart valves, aorta, and pulmonary artery, and lowest expression in the ventricles. Both RT-PCR methods led to similar results; however, the down-regulation of Chm-1 expression in groups III, IV and V compared to controls was more pronounced in the real-time RT-PCR experiments (89 +/- 28% versus 40 +/- 28%; 63 +/- 22% versus 29 +/- 19%; and 51 +/- 11% versus 13 +/- 7.4%; Chm-1/GAPDH ratio levels relative to the control group in conventional versus real-time RT-PCRs in groups III, IV and V, respectively). Only the decrease in group V was significantly different from group I with both methods (p < 0.05 and p = 0.001). Bacteremia alone resulted only in minor changes compared to controls. CONCLUSION: It is concluded that valvular Chm-1 expression is down-regulated in the early phase of IE, which is likely to promote leaflet vascularization and progression of the disease.

A rat model of cardiopulmonary bypass with cardioplegic arrest and hemodynamic assessment by conductance catheter technique.

BACKGROUND: Cardiopulmonary bypass (CPB) is known to induce systemic inflammation and cardiac dysfunction associated with a significant morbidity. Aim of the study was to develop an in vivo model of rat CPB with hypothermic cardiac arrest and the use of cardioplegia. MATERIAL AND METHODS: The CPB circuit consisted of a venous reservoir, membrane oxygenator, heat exchanger, and roller pump. CPB was instituted in adult male Wistar rats (400-500 g) for 60 min at a flow rate of 120 ml x kg(-1) x min(-1), including 15 min cooling to 32 degrees C, 30 min cardiac arrest with the use of cold crystalloid cardioplegia after aortic cross clamping, and 15 min of reperfusion and rewarming to 37 degrees C. Arterial blood pressure (MAP) and heart rate (HR) were monitored, arterial blood samples were analyzed. Left ventricular (LV) function parameters were assessed by intraventricular conductance catheter. Important technical aspects are: ventilation is required during partial bypass; anticoagulation should be performed immediately prior to CPB to reduce blood loss; active suction on venous drainage allows higher pump flows; and the small priming volume of the extracorporeal circuit (8 ml) avoids the need for donor blood. RESULTS: MAP remained stable prior to and during CPB.MAP and HR were significantly decreased 60 min after weaning from bypass. Hct was significantly lowered after hemodilution, but remained stable during CPB and 60 min after weaning from bypass. BE and pH remained stable throughout the experiment.Without inotropic support diastolic and systolic LV function parameters were impaired after 30 min of cardioplegic arrest followed by 15 min of reperfusion. Myocardial TNF-alpha mRNA levels were slightly increased (1.28-fold, p = 0.71), and IL-6 mRNA was significantly increased in the cardioplegia group (90.3-fold, p = 0.001). Both IL-6 and TNF-alpha plasma levels were significantly elevated in the cardioplegia group (TNF-alpha: 4.6-fold increase,p < 0.05; IL-6: 426.8-fold increase, p < 0.001). CONCLUSIONS: We have developed a rat CPB with mild hypothermic cardioplegic arrest. This rodent model is suitable to study clinically relevant problems related to CPB,myocardial protection and systemic inflammation.

Factor V Leiden does not affect bleeding in aprotinin recipients after cardiopulmonary bypass.

BACKGROUND: Carriers of the factor V Leiden mutation (FVL) are resistant to activated protein C proteolysis. Therefore, they are at increased risk of thromboembolic events. Aprotinin is an unspecific proteinase inhibitor frequently used during cardiac surgery procedures to reduce bleeding. However, aprotinin may cause thromboembolic complications after cardiopulmonary bypass (CPB). The primary endpoint of this study was the amount of blood loss after CPB in aprotinin recipients, and secondary endpoints were thromboembolic complications. METHODS: A total of 1,447 consecutive patients who underwent cardiac surgery with CPB were prospectively enrolled. All patients were screened for FVL by a fluorescence-based polymerase chain reaction method. Linear and logistic regression analyses were performed to assess associations of FVL on bleeding and thromboembolic complications. RESULTS: One hundred seven individuals (7.4%) were heterozygous FVL carriers. No difference was found between FVL carriers and noncarriers regarding age, sex, CPB, type of operation, EuroSCORE, antiplatelet treatment, and reoperation. FVL was not significantly associated with postoperative blood loss, whereas a significant influence was found for female sex (P < 0.0001), duration of CPB (P < 0.0001), reoperation (P = 0.001), and preoperative antiplatelet treatment (P < 0.002). Multiple linear regression analysis for total blood loss had an observed power of at least 99%. FVL carriers faced the same risk for postoperative transfusion (P = 0.391), reoperation (P = 0.675), myocardial infarction (P = 0.44), stroke (P = 0.701), and 30-day mortality (P = 0.4) as did noncarriers. CONCLUSIONS: These data suggest that FVL carriers do not have reduced blood loss compared with noncarriers. Furthermore, the combination of aprotinin and FVL does not enhance the risk for thromboembolic complications.

Characteristic of spontaneous transient outward potassium currents in vascular smooth muscle cells of porcine coronary artery.

Spontaneous transient outward currents (STOCs) play an important role in the myogenic regulation of small artery tone, such as coronary artery. In the present study, we investigated the electrophysiological properties and the regulation of STOCs in vascular smooth muscle cells (VSMCs) of porcine coronary artery by perforated patch-clamp technique. Our data showed that STOCs were dependent on voltage and extracellular calcium and they were highly variable in amplitudes and frequencies. STOCs superimposed stochastically onto whole-cell K(+) currents induced by step and ramp protocols. STOCs were completely abolished by ChTX [inhibitor of large-conductance Ca(2+)-activated potassium (BK(Ca)) channels], removal of extracellular Ca(2+), or addition of ryanodine (50 mumol/L) respectively. In contrast, CdCl2 and verapamil, inhibitors of voltage-dependent L-type Ca(2+) channels, had little effect on STOCs. Caffeine (5 mmol/L) transiently increased STOCs (hump), followed by a temporary inhibition. Ca(2+) ionophore A23187 increased both amplitude and frequency of STOCs. Na(+) ionophore monensin increased the frequency of STOCs. STOCs were strongly inhibited by KB-R7943, a selective inhibitor of the reverse mode of the Na(+)/Ca(2+) exchanger. Based on these observations, we conclude that STOCs are mediated by BK(Ca) channels. The generation and activation of STOCs depend upon Ca(2+) influx through Na(+)/Ca(2+) exchange and release of Ca(2+) from sarcoplasmic reticulum (SR) via ryanodine receptors. This suggests that Na(+)/Ca(2+) exchange determines calcium store refilling. Recycling of entering Ca(2+) from superficial SR may locally elevate Ca(2+) concentration at the plasma membrane, thereby activating BK(Ca) channels and then initiating STOCs.

Graft function, histopathology and morphometry of radial arteries used as conduits for myocardial revascularization in patients beyond age 70.

BACKGROUND: Coronary artery bypass grafting (CABG) using radial arteries has become a standard procedure for younger patients in many centers. However, advanced atherosclerotic changes may limit its use in older patients. We studied the effects of age on morphologic and histopathologic findings in CABG patients aged 70 years and older. METHODS: In 31 consecutive patients aged 70-85 (mean 77+/-8 years) scheduled for elective CABG, the left or right radial artery (RA) was used to graft the second target vessel (first graft: LIMA-LAD). Preoperative Doppler flow and Allen's test were satisfactory. Intraoperatively the distal segment adjacent to the anastomosis site of the RA was collected for histologic evaluation. Intraoperative angiography, measurement of flow, and resistance index (PI) were performed to document graft quality. Histopathology and morphometry were used to measure intimal and medial areas (IA, MA; microm2), intimal thickening index (ITI), relation between intimal and medial width at maximum intimal thickness (IMR), and percentage of luminal narrowing (%LN). RESULTS: The RA showed no evidence for stenosis of the conduits or the anastomosis. In all grafts flow and PI were satisfactory (76+/-14 ml/min; PI: 2.2+/-0.9). Histopathology and morphometry showed atherosclerotic changes in all RA grafts: IA: 890+/-971 (range 286-5244), MA: 2751+/-818 (range 1357-4989), ITI: 0.26+/-0.09 (range 0.12-0.44), IMR: 0.59+/-0.28 (range 0.21-1.13) %LN: 38+/-5 (range 13.2-61.7). Age as well as classic risk factors including diabetes, hypertension, smoking, and hyperlipidemia did not correlate with RA atherosclerosis. CONCLUSIONS: Excellent macroscopic and angiographic results were obtained. All grafts used showed minor to moderate atherosclerotic changes without severely altered indices of intimal thickening or luminal narrowing. The radial artery must be used with caution; however, age should not be an exclusion criterion per se.

Calcineurin and matrix protein expression in cardiac hypertrophy: evidence for calcineurin B to control excessive hypertrophic signaling.

In the compensatory state of human left ventricular hypertrophy (LVH), the remodeling processes in the extracellular matrix and the role of calcineurin (Cn) are not completely understood. The present work aimed to analyze the expression and activity of matrix metalloproteinases (MMPs), their endogenous inhibitors (TIMPs), and of Cn in patients with compensated LVH. By semiquantitative RT-PCR, Western blotting, and gelatine zymography, we determined mRNA, protein, and/or enzyme activity levels of MMPs, TIMPs, atrial natriuretic peptide (ANP), Cn subunits, and of the modulatory calcineurin-interacting protein (MCIP) 1. Myocardial samples from patients showing severe aortic stenosis, normal ejection fraction, and compensated LVH were compared with autopsy samples from healthy hearts. LVH patients showed upregulation of CnA-beta mRNA but downregulation of both CnB-alpha mRNA and protein. Total Cn activity (as determined through NF-AT phosphorylation and MCIP1 mRNA expression) was unchanged. There were no differences in gene expression and activities of MMP-2, MMP-9, and of TIMPs 1-4 between LVH patients and controls. As expected, ANP mRNA expression was high in LVH patients. We propose a prominent role for CnB in controlling Cn activity in compensated LVH. At this stage of the disease, MMP and TIMP activities are balanced.

Transcriptional regulation of alpha5beta1 integrin, fibronectin, VCAM-1, MCSF-1/c-fms, and MCP-1 in atrioventricular valves after valvular surgery and Staphylococcus aureus bacteremia.

OBJECTIVE: The primary event in the development of infective endocarditis (IE) is bacterial adherence to damaged heart valves. This includes capture, adhesion and internalization of bacteria into host cells. METHODS: We determined in an experimental rabbit model for IE whether a transient bacteremia caused by Staphylococcus aureus and/or endothelial heart valve lesions induce transcriptional regulation of alpha(5)beta(1) integrin, fibronectin, vascular cell adhesion molecule-1 (VCAM-1), macrophage colony-stimulating factor-1 (MCSF-1), c-fms proto-oncogene (MCSF-1 receptor), and macrophage chemoattractant protein-1 (MCP-1) in mitral and tricuspid valves. RESULTS: No significant upregulation was found after isolated bacteremia. Six hours after surgical manipulation valvular endothelial lesions led to a distinct modulation in the mRNA expression of proinflammatory cytokines (MCSF-1 and MCP-1), VCAM-1 and alpha(5)beta(1) integrin. The most evident differences between the mitral and tricuspid valves were seen in the significant upregulation of VCAM-1 mRNA on the tricuspid valve in the surgical groups, whereas there was no effect on the mitral valve. MCSF-1 and MCP-1 were dramatically upregulated in both valves after surgery. CONCLUSIONS: During the host defence mechanisms in the development of IE proinflammatory cytokines, cellular adhesion molecules, and molecules of the fibronectin/integrin axis are activated, showing distinct differences in right- and left-sided heart valves.

Atrial fibrosis in heart surgery patients Decreased collagen III/I ratio in postoperative atrial fibrillation.

In chronic atrial fibrillation, increased expression of angiotensinconverting enzyme (ACE) promotes upregulation of profibrotic proteins. Atrial fibrillation early after cardiac surgery (poAF) is common but unpredictable, and is regarded as a different entity. Therefore, the present study tested whether atrial expression of ACE, osteopontin (OPN), and collagen is elevated in patients with no history of AF but who develop poAF. Thus, 19 patients (66 +/- 9 years) with postoperative sinus rhythm (poSR) were compared to 14 patients (68 +/- 10) who experienced poAF. mRNA and protein expression were determined by RT-PCR and Western blotting. Picrosirius red was used to stain collagen. The medians for ACE, OPN, type I collagen (Col I), and type III collagen (Col III) mRNA and protein expression did not significantly differ between poSR and poAF (U-test). However, the Col III/I protein ratio was significantly lower in patients with poAF (2.62 vs. 1.09; poSR vs. poAF; p < 0.05). Our data suggest that the occurrence of poAF is not dependent on increased ACE and OPN expression, rendering the determination of preoperative OPN plasma levels inadequate to estimate the risk for the occurrence of poAF. Contrarily, a shift in atrial collagen expression levels in favor of Col I is linked to the occurrence of poAF.