Age-Dependent Effect of Pediatric Cardiac Progenitor Cells After Juvenile Heart Failure.

UNLABELLED: Children with congenital heart diseases have increased morbidity and mortality, despite various surgical treatments, therefore warranting better treatment strategies. Here we investigate the role of age of human pediatric cardiac progenitor cells (hCPCs) on ventricular remodeling in a model of juvenile heart failure. hCPCs isolated from children undergoing reconstructive surgeries were divided into 3 groups based on age: neonate (1 day to 1 month), infant (1 month to 1 year), and child (1 to 5 years). Adolescent athymic rats were subjected to sham or pulmonary artery banding surgery to generate a model of right ventricular (RV) heart failure. Two weeks after surgery, hCPCs were injected in RV musculature noninvasively. Analysis of cardiac function 4 weeks post-transplantation demonstrated significantly increased tricuspid annular plane systolic excursion and RV ejection fraction and significantly decreased wall thickness and fibrosis in rats transplanted with neonatal hCPCs compared with saline-injected rats. Computational modeling and systems biology analysis were performed on arrays and gave insights into potential mechanisms at the microRNA and gene level. Mechanisms including migration and proliferation assays, as suggested by computational modeling, showed improved chemotactic and proliferative capacity of neonatal hCPCs compared with infant/child hCPCs. In vivo immunostaining further suggested increased recruitment of stem cell antigen 1-positive cells in the right ventricle. This is the first study to assess the role of hCPC age in juvenile RV heart failure. Interestingly, the reparative potential of hCPCs is age-dependent, with neonatal hCPCs exerting the maximum beneficial effect compared with infant and child hCPCs. SIGNIFICANCE: Stem cell therapy for children with congenital heart defects is moving forward, with several completed and ongoing clinical trials. Although there are studies showing how children differ from adults, few focus on the differences among children. This study using human cardiac progenitor cells shows age-related changes in the reparative ability of cells in a model of pediatric heart failure and uses computational and systems biology to elucidate potential mechanisms.

Fibrin Network Changes in Neonates after Cardiopulmonary Bypass.

BACKGROUND: Quantitative and qualitative differences in the hemostatic systems exist between neonates and adults, including the presence of "fetal" fibrinogen, a qualitatively dysfunctional form of fibrinogen that exists until 1 yr of age. The consequences of "fetal" fibrinogen on clot structure in neonates, particularly in the context of surgery-associated bleeding, have not been well characterized. Here, the authors examine the sequential changes in clotting components and resultant clot structure in a small sample of neonates undergoing cardiac surgery and cardiopulmonary bypass (CPB). METHODS: Blood samples were collected from neonates (n = 10) before surgery, immediately after CPB, and after the transfusion of cryoprecipitate (i.e., adult fibrinogen component). Clots were formed from patient samples or purified neonatal and adult fibrinogen. Clot structure was analyzed using confocal microscopy. RESULTS: Clots formed from plasma obtained after CPB and after transfusion were more porous than baseline clots. Analysis of clots formed from purified neonatal and adult fibrinogen demonstrated that at equivalent fibrinogen concentrations, neonatal clots lack three-dimensional structure, whereas adult clots were denser with significant three-dimensional structure. Clots formed from a combination of purified neonatal and adult fibrinogen were less homogenous than those formed from either purified adult or neonatal fibrinogen. CONCLUSIONS: The results of this study confirm that significant differences exist in clot structure between neonates and adults and that neonatal and adult fibrinogen may not integrate well. These findings suggest that differential treatment strategies for neonates should be pursued to reduce the demonstrated morbidity of blood product transfusion.

Optimizing Thrombin Generation with 4-Factor Prothrombin Complex Concentrates in Neonatal Plasma After Cardiopulmonary Bypass.

BACKGROUND: Bleeding is a serious complication after pediatric cardiopulmonary bypass (CPB) that is associated with an increase in perioperative morbidity and mortality. Four-factor prothrombin complex concentrates (4F-PCCs) have been used off-label to supplement transfusion protocols for bleeding after CPB in adults; however, data on their use in neonates are limited. In this study, we hypothesized that 4F-PCCs administered ex vivo to neonatal plasma after CPB will increase thrombin generation. METHODS: Fifteen neonates undergoing complex cardiac repairs requiring CPB were enrolled in this prospective study. Arterial blood was obtained after anesthesia induction but before CPB (baseline), after CPB following heparin reversal, and after our standardized transfusion of a quarter of a platelet apheresis unit (approximately 20 mL·kg) and 3 units of cryoprecipitate. Kcentra (CSL Behring), a 4F-PCC with nonactivated factor VII (FVII), and factor 8 inhibitor bypassing activity (FEIBA; Baxter Healthcare Corporation), a 4F-PCC with activated FVII, were added ex vivo to plasma obtained after CPB to yield concentrations of 0.1 and 0.3 IU·mL. Calibrated automated thrombography was used to determine thrombin generation for each sample. RESULTS: The addition of Kcentra to plasma obtained after CPB resulted in a dose-dependent increase in the median (99% confidence interval) peak amount of thrombin generation (42.0 [28.7-50.7] nM for Kcentra 0.1 IU·mL and 113.9 [99.0-142.1] nM for Kcentra 0.3 IU·mL). The rate of thrombin generation was also increased (15.4 [6.5-24.6] nM·min for Kcentra 0.1 IU·mL and 48.6 [29.9-66.6] nM·min for Kcentra 0.3 IU·mL). The same was true for FEIBA (increase in peak: 39.8 [27.5-49.2] nM for FEIBA 0.1 IU·mL and 104.6 [92.7-124.4] nM for FEIBA 0.3 IU·mL; increase in rate: 17.4 [7.4-28.8] nM·min FEIBA 0.1 IU·mL and 50.5 [26.7- 63.1] nM·min FEIBA 0.3 IU·mL). In the posttransfusion samples, there was a significant increase with Kcentra in the median (99% confidence interval) peak amount (41.1 [21.0-59.7] nM for Kcentra 0.1 IU·mL and 126.8 [106.6- 137.9] nM for Kcentra 0.3 IU·mL) and rate (18.1 [-6.2 to 29.2] nM·min for Kcentra 0.1 IU·mL and 53.2 [28.2-83.1] nM·min for Kcentra 0.3 IU·mL) of thrombin generation. Again, the results were similar for FEIBA (increase in peak: 43.0 [36.4-56.7] nM for FEIBA 0.1 IU·mL and 109.2 [90.3-136.1] nM for FEIBA 0.3 IU·mL; increase in rate: 25.0 [9.1-32.6] nM·min for FEIBA 0.1 IU·mL and 59.7 [38.5-68.7] nM·min for FEIBA 0.3 IU·mL). However, FEIBA produced in a greater median reduction in lag time of thrombin generation versus Kcentra in samples obtained after CPB (P = 0.003 and P = 0.0002 for FEIBA versus Kcentra at 0.1 and 0.3 IU·mL, respectively) and in samples obtained after transfusion (P < 0.0001 for FEIBA versus Kcentra at 0.1 and 0.3 IU·mL). CONCLUSIONS: After CPB, thrombin generation in neonatal plasma was augmented by the addition of 4F-PCCs. The peak amount and rate of thrombin generation were enhanced in all conditions, whereas the lag time was shortened more with FEIBA. Our findings suggest that the use of 4F-PCCs containing activated FVII may be an effective adjunct to the initial transfusion of platelets and cryoprecipitate to augment coagulation and control bleeding in neonates after CPB.

Correlations between activated clotting time values and heparin concentration measurements in young infants undergoing cardiopulmonary bypass.

BACKGROUND: Monitoring heparin concentration along with the activated clotting time (ACT) may provide a more accurate guide for the administration of heparin to infants during cardiopulmonary bypass (CPB). However, standard laboratory assays of heparin concentration (antifactor Xa heparin concentration) require plasma instead of whole blood, and results are not immediately available to clinicians. Alternatively, measurements of whole blood heparin concentration may be performed at the bedside using an automated protamine titration device, the Hepcon instrument (Hepcon Hemostasis Management System Plus; Medtronics, Minneapolis, MN). The purpose of this investigation was to compare ACT measurements from 3 commercially available instruments and bedside measurements of whole blood heparin concentration using the Hepcon instrument with laboratory measurements of antifactor Xa plasma heparin concentration in infants younger than 6 months of age undergoing CPB. METHODS: Forty-four pediatric patients younger than 6 months of age scheduled for elective cardiac surgery requiring CPB were enrolled in this prospective study. Blood samples were drawn 3 minutes after the initial heparin bolus and immediately before the termination of CPB to obtain measurements of heparin anticoagulation. Kaolin-activated ACTs were performed with the Hemochron (International Technidyne Corporation, Edison, NJ), Hepcon, and i-STAT (i-STAT Corporation, East Windsor, NJ) instruments. Whole blood heparin concentration was measured using the Hepcon instrument. Plasma heparin concentration was measured using an antifactor Xa chromogenic substrate assay. RESULTS: Immediately after the initial heparin bolus, none of the ACT values correlated with plasma heparin concentration. When measured immediately before the termination of CPB, only the i-STAT ACT showed a moderate correlation. Conversely, bedside measurements of whole blood heparin concentration showed satisfactory agreement with laboratory measurements of plasma heparin concentration at both time points (concordance correlation coefficients 0.30 and 0.67, respectively). There is a bias in that antifactor Xa-measured plasma heparin concentration tends to be higher than Hepcon-measured whole blood heparin concentration. CONCLUSIONS: In infants younger than 6 months old undergoing CPB, caution is warranted when using ACT values as the sole indication of adequate heparin anticoagulation. In general, ACT prolongation correlates poorly with plasma heparin concentration. Only i-STAT ACT values showed a moderate correlation when measured immediately before the termination of CPB. Alternatively, bedside measurements of whole blood heparin concentration measured by the Hepcon instrument agreed well with antifactor Xa laboratory measurements. Our data support the clinical utility of bedside measurements of heparin concentration to provide timely, convenient, and accurate measurements of heparin concentration in these infants.

Use of recombinant factor VIIa for uncontrolled bleeding in neonates after cardiopulmonary bypass.

BACKGROUND: Increasingly, recombinant activated factor VII (rFVIIa) is used adjunctively in nonhemophiliacs to control hemorrhage unresponsive to conventional therapy in a variety of settings including postcardiopulmonary bypass (CPB). Studies examining rFVIIa administration to neonates after CPB are limited. The goal of this study was to evaluate retrospectively the clinical outcomes of neonates treated at our institution with rFVIIa for uncontrolled post-CPB bleeding. METHODS: We retrospectively identified eight neonates undergoing complex congenital cardiac surgery who received rFVIIa, either intraoperatively or postoperatively, for uncontrolled post-CPB bleeding. Transfusion trends and prothrombin times (PT) were assessed both pre- and post-rFVIIa administration. Chest tube drainage volumes were recorded pre- and post-rFVIIa administration in those neonates receiving rFVIIa postoperatively in the intensive care unit. We documented such adverse events as thrombosis, dialysis (hemodialysis and peritoneal dialysis), extracorporeal membrane oxygenation (ECMO) and in-hospital mortality. RESULTS: The mean amount of transfused packed red blood cells, platelets and fresh frozen plasma decreased significantly after the administration of rFVIIa. Transfusion of cryoprecipitate trended towards a decrease but did not reach statistical significance. PT values also decreased significantly after the administration of rFVIIa. A high mortality was found in neonates exposed to both rFVIIa and ECMO; however, this was not significantly different from the mortality of neonates exposed to ECMO alone. CONCLUSIONS: Administration of rFVIIa to neonates for the treatment of uncontrolled post-CPB bleeding significantly reduced transfusion requirements and normalized PT values. Future randomized, controlled trials are needed to evaluate the potential hemostatic benefit and adverse effects of rFVIIa administration to neonates following CPB.

Force frequency relationship of the human ventricle increases during early postnatal development.

Understanding developmental changes in contractility is critical to improving therapies for young cardiac patients. Isometric developed force was measured in human ventricular muscle strips from two age groups: newborns (<2 wk) and infants (3-14 mo) undergoing repair for congenital heart defects. Muscle strips were paced at several cycle lengths (CLs) to determine the force frequency response (FFR). Changes in Na/Ca exchanger (NCX), sarcoplasmic reticulum Ca-ATPase (SERCA), and phospholamban (PLB) were characterized. At CL 2000 ms, developed force was similar in the two groups. Decreasing CL increased developed force in the infant group to 131 +/- 8% (CL 1000 ms) and 157 +/- 18% (CL 500 ms) demonstrating a positive FFR. The FFR in the newborn group was flat. NCX mRNA and protein levels were significantly larger in the newborn than infant group whereas SERCA levels were unchanged. PLB mRNA levels and PLB/SERCA ratio increased with age. Immunostaining for NCX in isolated newborn cells showed peripheral staining. In infant cells, NCX was also found in T-tubules. SERCA staining was regular and striated in both groups. This study shows for the first time that the newborn human ventricle has a flat FFR, which increases with age and may be caused by developmental changes in calcium handling.