Antonella Galeone

Aortic valvular interstitial cells apoptosis and calcification are mediated by TNF-related apoptosis-inducing ligand

BACKGROUND/OBJECTIVES Calcific aortic valvular disease (CAVD) is an actively regulated process characterized by the activation of specific osteogenic signaling pathways and apoptosis. We evaluated the involvement in CAVD of the TNF-related apoptosis-inducing ligand (TRAIL), an apoptotic molecule which induces apoptosis by interacting with the death receptor (DR)-4 and DR5, and whose activity is modulated by the decoy receptor (DcR)-1 and DcR2. METHODS Sections of calcific and normal aortic valves, obtained at surgery time, were subjected to immunohistochemistry and confocal microscopy for TRAIL immunostaining. Valvular interstitial cells (VICs) isolated from calcific (C-VICs) and normal (N-VICs) aortic valves were investigated for the gene and protein expression of TRAIL receptors. Cell viability was assayed by MTT. Von Kossa staining was performed to verify C-VIC ability to produce mineralized nodules. TRAIL serum levels were detected by ELISA. RESULTS Higher levels of TRAIL were detected in calcific aortic valves and in sera from the same patients respect to controls. C-VICs express significantly higher mRNA and protein levels of DR4, DR5, DcR1, DcR2 and Runx2 compared to N-VICs. C-VICs and N-VICs, cultured in osteogenic medium, express significantly higher mRNA levels of DR4, Runx2 and Osteocalcin compared to baseline. C-VICs and N-VICs were sensitive to TRAIL-apoptotic effect at baseline and after osteogenic differentiation, as demonstrated by MTT assay and caspase-3 activation. TRAIL enhanced mineralized matrix nodule synthesis by C-VICs cultured in osteogenic medium. CONCLUSIONS TRAIL is characteristically present within calcific aortic valves, and mediates the calcification of aortic valve interstitial cells in culture through mechanism involving apoptosis.

The Role of TNF-α and TNF Superfamily Members in the Pathogenesis of Calcific Aortic Valvular Disease

Calcific aortic valve disease (CAVD) represents a slowly progressive pathologic process associated with major morbidity and mortality. The process is characterized by multiple steps: inflammation, fibrosis, and calcification. Numerous studies focalized on its physiopathology highlighting different “actors” for the multiple “acts.” This paper focuses on the role of the tumor necrosis factor superfamily (TNFSF) members in the pathogenesis of CAVD. In particular, we discuss the clinical and experimental studies providing evidence of the involvement of tumor necrosis factor-alpha (TNF-α), receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL), its membrane receptor RANK and its decoy receptor osteoprotegerin (OPG), and TNF-related apoptosis-inducing ligand (TRAIL) in valvular calcification.

Monitoring incomplete heparin reversal and heparin rebound after cardiac surgery.

OBJECTIVES To assess the incidence of incomplete heparin reversal and heparin rebound after cardiac surgery with cardiopulmonary bypass (CPB) and the ability of the activated coagulation time (ACT) and thromboelastography (TEG) to detect these phenomena. DESIGN Prospective single-center study. SETTING University hospital. PARTICIPANTS Forty-one patients undergoing elective cardiac surgery with CPB and with normal preoperative TEG parameters. INTERVENTIONS ACT, TEG, and plasma heparin levels were measured in all patients at 5 different times between 20 minutes and 3 hours after protamine administration. The variability of TEG reaction time (R) with and without heparinase (delta-R [DR]) was used to detect the presence of residual heparin. MEASUREMENTS AND MAIN RESULTS Plasma heparin expressed as anti-FXa activity was detected in 180 (88%) samples. At univariate analysis, ACT, R-kaolin (R-k), and DR significantly correlated with plasma heparin concentration (respectively, p = 0.007, p = 0.006, and p = 0.002). At multivariate analysis, R-k and DR remained associated with plasma heparin concentration (respectively, p = 0.014 and p = 0.004). Greater quartiles of heparin were associated with higher values of R-k and DR. Combined procedures had significantly lower DR than isolated procedures (p = 0.017), and CPB time and heparinization time positively correlated with R-k (respectively, p = 0.044 and p = 0.022). No association was observed between heparin concentration, ACT, and TEG parameters with postoperative bleeding and need for blood and blood components transfusions. CONCLUSIONS Heparin rebound and incomplete heparin reversal are very common phenomena after cardiac surgery with CPB; ACT is not able to detect residual heparin activity, whereas TEG analysis with and without heparinase allows the diagnosis of heparin rebound.

Formation of anti-platelet factor 4/heparin antibodies after cardiac surgery: Influence of perioperative platelet activation, the inflammatory response, and histocompatibility leukocyte antigen status

BACKGROUND Anticoagulation therapy with heparin induces antibodies that recognize multimolecular complexes of platelet factor 4 bound to heparin (anti-platelet factor 4/heparin antibodies). Considering that cardiac surgery induces an intense platelet activation and proinflammatory response, we examined the relationship between formation of anti-platelet factor 4/heparin antibodies and plasma levels of platelet factor 4 and interleukin 6. We also examined the relationship between anti-platelet factor 4/heparin seroconversion and the histocompatibility leukocyte antigen system. METHODS In 71 patients undergoing cardiac surgery, anti-platelet factor 4/heparin antibody levels were evaluated by means of enzyme-linked immunosorbent assay preoperatively and 14 days postoperatively. Platelet serotonin release assays were performed to assess the platelet-activating potential of the antibodies. Plasma levels of platelet factor 4 and interleukin 6 were assayed at prespecified time points. Histocompatibility leukocyte antigen status was assessed preoperatively in all patients and was compared with that of 6156 healthy subjects. RESULTS Thirty-seven (52%) patients had anti-platelet factor 4/heparin antibodies with an OD value of 0.45 or greater in 1 or more of the assays. Applying strict seroconversion criteria (>2-fold increase in Optical Density), only 16 (22.5%) patients had evidence of anti-platelet factor 4/heparin antibody seroconversion after the operation. Neither the presence of anti-platelet factor 4/heparin antibodies nor seroconversion influenced postoperative outcomes. The CW4 allele was significantly more frequent among seroconverted patients (46.9% vs 19.1%, P = .002). Platelet factor 4 levels did not influence seroconversion. Patients with anti-platelet factor 4/heparin levels of 0.45 OD units or greater 14 days after the operation had significantly higher interleukin 6 levels measured 1 hour after protamine administration. DISCUSSION Patients with a greater amount of perioperative inflammation could be more likely to have anti-platelet factor 4/heparin antibodies 1 to 2 weeks later. We provide additional evidence that the histocompatibility leukocyte antigen CW4 confers genetic susceptibility in an acquired inflammatory disorder that includes the anti-platelet factor 4/heparin immune response.

Coagulation-Fibrinolysis Changes During Off-Pump Bypass: Effect of Two Heparin Doses

BACKGROUND To date, no study has tested the effect of different heparin dosages on the hemostatic changes during off-pump coronary artery bypass graft (OPCABG) surgery, and a wide variety of empirical anticoagulation protocols are being applied. We tested the effect of two different heparin dosages on the activation of the hemostatic system in patients undergoing OPCABG procedures. METHODS Forty-two patients eligible for OPCABG procedures were assigned in a randomized fashion to low-dose heparin (150 IU/kg) or high-dose heparin (300 IU/kg). Prothrombin fragment 1+2, plasmin/alpha(2)-plasmin inhibitor complex, D-dimer, soluble tissue factor, tissue factor pathway inhibitor, total thrombin activatable fibrinolysis inhibitor (TAFI), and activated TAFIa were assayed by specific enzyme-linked immunosorbent assays at six different timepoints, before, during, and after surgery. Platelet function was evaluated by means of an in vitro bleeding time test, platelet function analyzer-100. RESULTS The OPCABG surgery was accompanied by significant changes of all plasma biomarkers, indicative of systemic activation of coagulation and fibrinolysis. A significant increase in circulating TAFIa was detected perioperatively and postoperatively, and multiple regression analysis indicated that prothrombin F1+2 but not plasmin/alpha(2)-antiplasmin complex was independently associated with TAFIa level. Platelet function analyzer-100 values did not change significantly after OPCABG. All hemostatic changes were similar in the two heparin groups, even perioperatively, when the difference in anticoagulation was maximal. CONCLUSIONS Both early and late hemostatic changes, including TAFI activation, are similarly affected in the low-dose and high-dose heparin groups, suggesting that the increase in heparin dosage is not accompanied by a better control of clotting activation during OPCABG surgery.

Activation of the receptor activator of the nuclear factor-κB ligand pathway during coronary bypass surgery: comparison between on- and off-pump coronary artery bypass surgery procedures

OBJECTIVES The receptor activator of the nuclear factor kappa-B (NF-κB) ligand (RANKL), its membrane receptor RANK and its decoy receptor osteoprotegerin (OPG) are all members of the tumour necrosis factor family involved in bone metabolism and immune response. We evaluated the activation of the OPG/RANKL/RANK pathway in patients undergoing cardiac surgery with and without cardiopulmonary bypass (CPB). METHODS Twenty consecutive patients undergoing elective coronary artery surgery were enrolled in the study and assigned either to the on-pump or to the off-pump group. Pre- and postoperative serum levels of OPG and RANKL were evaluated by enzyme-linked immunosorbent assay; gene expression of OPG, RANKL, RANK and NF-κB p50 subunits were determined by real-time polymerase chain reaction in peripheral blood T-cells and monocytes. RESULTS Serum levels of OPG significantly increased after surgery in both groups, whereas serum levels of RANKL did not differ over time. T-cells from the on-pump group showed increased gene expression of OPG, RANKL and RANK after the intervention, whereas no mRNA variation for these genes was detected in T-cells from off-pump patients. Gene expression of p50 subunit increased in T-cells and monocytes from both groups. CONCLUSIONS Cardiac surgery induces the activation of the OPG/RANKL/RANK pathway; both on- and off-pump procedures are associated with increased postoperative OPG serum levels and up-regulation of the NF-κB p50 subunit.

Early onset of heparin-induced thrombocytopenia with thrombosis after open heart surgery: importance of an early diagnosis and Lepirudin treatment.

Heparin-induced thrombocytopenia with thrombosis (HITT) is a rare complication of cardiac surgery with cardiopulmonary bypass. We report two cases of HITT treated with the direct thrombin inhibitor Lepirudin. Immediate diagnosis was essential to prompt heparin discontinuation and successful early Lepirudin administration in the first case. In the second, the presence of an intra-aortic balloon pump delayed HITT recognition, and Lepirudin infusion could not prevent limb amputation. In both cases HITT occurred earlier (< 5 days after heparin exposure) than its usual presentation.

Performance of existing risk scores around heart transplantation: validation study in a 4-year cohort

Several risk scores exist to help identify best candidate recipients for heart transplantation (HTx). This study describes the performance of five heart failure risk scores and two post‐HTx mortality risk scores in a French single‐centre cohort. All patients listed for HTx through a 4‐year period were included. Waiting‐list risk scores [Heart Failure Survival Score (HFSS), Seattle Heart Failure Model (SHFM), Meta‐Analysis Global Group in Chronic Heart Failure (MAGGIC), Organized Program to Initiate Lifesaving Treatment in Hospitalized Patients with Heart Failure (OPTIMIZE‐HF) and Get With The Guidelines‐Heart Failure (GWTG‐HF)] and post‐HTx scores Index for Mortality Prediction After Cardiac Transplantation (IMPACT and CARRS) were computed. Main outcomes were 1‐year mortality on waiting list and after HTx. Performance was assessed using receiver operator characteristic (ROC), calibration and goodness‐of‐fit analyses. The cohort included 414 patients. Waiting‐list mortality was 14.0%, and post‐HTx mortality was 16.3% at 1‐year follow‐up. Heart failure risk scores had adequate discrimination regarding waiting‐list mortality (ROC AUC for HFSS = 0.68, SHFM = 0.74, OPTIMIZE‐HF = 0.72, MAGGIC = 0.70 and GWTG = 0.77; all P‐values <0.05). On the contrary, post‐HTx risk scores did not discriminate post‐HTx mortality (AUC for IMPACT = 0.58, and CARRS = 0.48, both P‐values >0.50). Subgroup analysis on patients undergoing HTx after ventricular assistance device (VAD) implantation (i.e. bridge‐to‐transplantation) (n = 36) showed an IMPACT AUC = 0.72 (P < 0.001). In this single‐centre cohort, existing heart failure risk scores were adequate to predict waiting‐list mortality. Post‐HTx mortality risk scores were not, except in the VAD subgroup.