Sirisha Emani

Cardiac extracellular matrix-fibrin hybrid scaffolds with tunable properties for cardiovascular tissue engineering.

Solubilized cardiac extracellular matrix (ECM) is being developed as an injectable therapeutic that offers promise for promoting cardiac repair. However, the ECM alone forms a hydrogel that is very soft compared to the native myocardium. As both the stiffness and composition of the ECM are important in regulating cell behavior and can have complex synergistic effects, we sought to develop an ECM-based scaffold with tunable biochemical and mechanical properties. We used solubilized rat cardiac ECM from two developmental stages (neonatal, adult) combined with fibrin hydrogels that were cross-linked with transglutaminase. We show that ECM was retained within the gels and that the Youngs modulus could be tuned to span the range of the developing and mature heart. C-kit+ cardiovascular progenitor cells from pediatric patients with congenital heart defects were seeded into the hybrid gels. Both the elastic modulus and composition of the scaffolds impacted the expression of endothelial and smooth muscle cell genes. Furthermore, we demonstrate that the hybrid gels are injectable, and thus have potential for minimally invasive therapies. ECM-fibrin hybrid scaffolds offer new opportunities for exploiting the effects of both composition and mechanical properties in directing cell behavior for tissue engineering.

Dendritic cells pulsed with pancreatic cancer total tumor RNA generate specific antipancreatic cancer T cells

RNA-based dendritic cell immunotherapy with the use of total tumor RNA provides the potential to generate a polyclonal immune response to multiple known and unknown tumor antigens without HLA restriction. Our study evaluated this approach as potential immunotherapy for patients with pancreatic cancer. Dendritic cells were generated using adherent monocytes isolated from peripheral blood of patients with pancreatic cancer and evaluated phenotypically by flow cytometry to determine whether dendritic cells could be generated from the blood of patients with pancreatic cancer. Immature dendritic cells were transfected with mRNA encoding full-length carcinoembryonic antigen (CEA) or pancreatic cancer total tumor messenger RNA, and then matured. Matured dendritic cell phenotypes were also analyzed by flow cytometry. Transfected, matured dendritic cells were used to stimulate autologous T cells, and the resultant antigen-specific effector T cells were analyzed by interferon-γ Elispot assay. Immature dendritic cells with characteristic phenotypic markers CD40, CD80, and CD86 were successfully isolated from the blood of patients with pancreatic cancer. Incubation with maturation agents increased expression of CD80 and CD83, demonstrating the induction of a mature antigen-presenting phenotype. Dendritic cells transfected with a pancreatic cancer-associated antigen (CEA) generated antigen-specific T cells (P < 0.05). Dendritic cells transfected with autologous total tumor pancreatic cancer RNA generated T cells that specifically recognized HLA-matched pancreatic cancer cell lines (P < 0.05 compared to control cell lines). Dendritic cells from patients with pancreatic cancer maintain the ability to translate and process transfected RNA and serve as mature antigen-presenting cells. These RNA-transfected dendritic cells from pancreatic cancer patients successfully generate specific T cells against the pancreatic cancer-associated antigen CEA as well as T cells that specifically recognize pancreatic cancer cells. These data suggest that total tumor RNA-pulsed dendritic cells may have potential as an adjuvant immunotherapy for patients with pancreatic cancer.

Increased vascular permeability after cardiopulmonary bypass in patients with diabetes is associated with increased expression of vascular endothelial growth factor and hepatocyte growth factor

BACKGROUNDnSeveral inflammatory mediators such as vascular endothelial growth factor and hepatocyte growth factor are known to play a critical role in the regulation of vascular permeability and angiogenesis. We studied the serum levels of growth factors and gene expression profiles of genes involved in growth factor signaling in the peripheral blood of patients with and patients without diabetes following cardiopulmonary bypass and cardioplegic arrest.nnnMETHODSnSerum and total RNA were obtained from the blood samples collected from patients with diabetes and matched patients without diabetes (n = 7 patients each) who had coronary artery bypass graft before and 6 hours and 4 days after cardiopulmonary bypass/cardioplegic arrest. The cytokine panel, consisting of growth factors such as vascular endothelial growth factor, hepatocyte growth factor, fibroblast growth factor, and epidermal growth factor, was quantified in patients with diabetes and patients without diabetes before and 6 hours and 4 days post-cardiopulmonary bypass/cardioplegic arrest using multiplex cytokine quantification system. cDNA microarray analysis was performed and fold-change was calculated.nnnRESULTSnLength of hospitalization (10 vs 6 days; P = .04) and weight gain (5 vs 2.5 kg; P = .001) were significantly greater for patients with diabetes compared with patients without diabetes. The serum levels of vascular endothelial growth factor and hepatocyte growth factor were significantly elevated in patients with diabetes when compared with patients without diabetes before versus 6 hours post-cardiopulmonary bypass/cardioplegic arrest. In addition, significantly elevated mRNA expression of hypoxia-inducible factor-1alpha, cyclic adenosine monophosphate response element binding protein, and E1A binding protein p300 (more than twofold) was observed 4 days post-cardiopulmonary bypass/cardioplegic arrest exclusively in patients with diabetes.nnnCONCLUSIONSnThe differential profile of gene and protein expression of growth factors and their related genes in patients with diabetes and patients without diabetes could be associated with increased edema and weight gain in patients with diabetes after cardiopulmonary bypass/cardioplegic arrest.

Hypercoagulability markers predict thrombosis in single ventricle neonates undergoing cardiac surgery.

BACKGROUNDnIncidence of thrombosis in neonates undergoing cardiac surgery is as high as 20%, and single ventricle physiology (SVP) may present an even higher risk. We hypothesize that SVP is a risk factor for thrombosis in neonates undergoing cardiac surgery, and hypercoagulability biomarkers are predictive of postoperative thrombosis.nnnMETHODSnRecords of 512 neonates undergoing cardiac surgery were retrospectively reviewed. Thrombosis was defined by clinical events (shunt thrombosis, limb ischemia, and stroke) or intravascular or cardiac thrombus by echocardiography. Clinical variables, including SVP and cardiopulmonary bypass (CPB), were analyzed using multivariable logistic regression. A hypercoagulability biomarker panel was obtained in a subset of patients with SVP and compared between neonates with and without thrombosis.nnnRESULTSnThrombosis was detected in 51 of 512 neonates undergoing cardiac surgery. Intensive care and hospital lengths of stay were longer in patients who experienced thrombosis compared with those who did not (14 ± 13 vs 6 ± 1 days, 23 ± 4 vs 13 ± 1 days, p < 0.001). The SVP and use of CPB were significant risk factors for thrombosis, and the rate of thrombosis in SVP patients was 16.2% (16 of 99) compared with 8.5% (35 of 413) in non-SVP patients (p = 0.038). Thrombin generation, plasminogen activator inhibitor, and thrombin activatable fibrinolysis inhibitor were significantly elevated in SVP patients with thrombosis compared to without thrombosis (p < 0.05).nnnCONCLUSIONSnSingle ventricle physiology patients are at higher risk for thrombosis compared with other neonates after cardiac surgery. Hypercoagulable panel testing may help risk stratify patients and guide patient specific anticoagulation management in the postoperative period.

Aspirin unresponsiveness predicts thrombosis in high-risk pediatric patients after cardiac surgery

OBJECTIVEnThrombosis occurs in up to 26% of patients with congenital heart disease after cardiac surgery and is associated with increased morbidity and mortality. Aspirin is commonly administered to reduce the risk of thrombosis, yet aspirin responsiveness is rarely assessed. In this study, we hypothesize that inadequate response to aspirin is associated with increased risk of thrombosis after selected congenital cardiac procedures considered to be high risk for thrombosis.nnnMETHODSnPatients undergoing high-risk congenital cardiac surgery who received postoperative aspirin (N = 95) were studied. Response to aspirin was determined using the VerifyNow system several days after administration. Patients were monitored prospectively for 30 days for the development of a thrombosis event and the relationship between aspirin unresponsiveness and a thrombosis event was determined by the Fisher exact test.nnnRESULTSnRate of aspirin unresponsiveness (≥550 aspirin reaction units [ARU]) was 10 of 95 (10.5%) and was highest in patients weighing less than 5 kg given 20.25 mg/d of aspirin. Thrombosis events occurred in 7 patients (7.4%). Thrombosis was observed in 6 of 10 (60%) patients who were unresponsive to aspirin, compared with 1 of 85 (1.2%) patients who were responsive to aspirin (P < .001). In 2 patients who were unresponsive to the initial aspirin dose, an increase in dose resulted in an adequate therapeutic aspirin response (ARU < 550), suggesting insufficiency rather than true resistance in a subset of patients.nnnCONCLUSIONSnPostoperative thrombosis is associated with aspirin unresponsiveness in this patient population. In high-risk patients, monitoring of aspirin therapy and consideration of dose adjustment or alternative agents for unresponsive patients may be justified and warrants further investigation in a prospective trial.

Hypercoagulability panel testing predicts thrombosis in neonates undergoing cardiac surgery

Thrombosis contributes to morbidity and mortality in neonates following cardiac surgery. Alterations in hemostatic factors following cardiac surgery have been described, but there is no data correlating these changes with risk of thrombosis in neonates. The aim of this study is to predict thrombosis in neonates undergoing cardiac surgery by assessment of a panel of hypercoagulability markers. Neonates undergoing cardiac surgery were enrolled preoperatively and prospectively followed. Preoperative hypercoagulability panel testing included thrombin generation assay (TGA), immunoassays for antithrombin III, protein C, protein S, factor VIII, thrombin‐activatable fibrinolytic inhibitor (TAFI), plasminogen activator inhibitor‐1 (PAI‐1), and cardiolipin antibody. Postoperative thrombosis was defined by clinical events (shunt thrombosis, limb ischemia, and stroke) or imaging (intravascular or intracardiac thrombus). Risk factors for thrombosis were assessed. One hundred neonates were enrolled in the study over a two‐year period. The incidence of postoperative in‐hospital thrombosis was 20%. The only significant clinical risk factor associated with thrombosis was the single ventricle physiology. Hypercoagulability factors associated with increased risk of thrombosis by univariate analysis were elevated PAI‐1, TAFI, and TGA, and presence of anticardiolipin antibodies. Multivariable logistic regression analysis demonstrated that elevated PAI‐1 (Pu2009=u20090.015), TAFI (Pu2009=u20090.028), and TGA (Pu2009=u20090.007) were independent predictors of thrombosis. Hypercoagulability panel testing may help identify neonates at high risk for thrombosis following cardiac surgery. Future studies are warranted to determine if high risk patients benefit from targeted anticoagulation therapies. Am. J. Hematol. 89:151–155, 2014.

RNA stability regulates differential expression of the metastasis protein, osteopontin, in hepatocellular cancer.

BACKGROUNDnOsteopontin (OPN) is a potential therapeutic target in hepatocellular carcinoma (HCC), because it is a critical mediator of metastatic function. The molecular mechanisms that determine expression of OPN in HCC, however, are unknown. In this study, we examine differential OPN expression in the 2 HCC cell lines: HepG2 and Hep3B.nnnMETHODSnOPN expression, metastatic function, OPN promoter activity, and active transcription of OPN mRNA and its decay were assessed in the 2 HCC cell lines using standard techniques. RNA gel-shift assays were performed to determine binding of cytoplasmic proteins to OPN mRNA.nnnRESULTSnExpression of OPN cellular/secreted protein and mRNA was greater in HepG2 than Hep3B cells (P < .01). Transient transfection of the OPN promoter construct demonstrated equivalent luciferase activities in the 2 cell lines; the rate of transcription was also equivalent as determined by chromatin immuno-precipitation assay. OPN mRNA half-life was 21 +/- 1 h and 3 +/- 1 h in HepG2 and Hep3B, respectively (P < .02). In HepG2 and Hep3B, the nucleotide sequence of OPN and its 5-UTR, 3-UTR, and poly (A) tail lengths were identical. A luciferase construct coupled in line with OPN-5-UTR and OPN 3-UTR presented greater expression in HepG2 (P < .01 vs Hep3B). Deletion of nt 10-57 of the OPN 5-UTR restored luciferase and HA-tagged OPN protein expression in Hep3B but not in Hep G2. RNA gel-shift assays demonstrate different patterns of protein binding to OPN 5-UTR between the 2 HCC cell lines.nnnCONCLUSIONSnWe conclude that RNA stability is a new, previously unrecognized mechanism that regulates OPN expression in HCC to convey metastatic function.

Platelet testing to guide aspirin dose adjustment in pediatric patients after cardiac surgery

Objectives: Thrombosis is associated with increased morbidity and mortality in pediatric patients undergoing cardiac surgery. Although aspirin commonly is used for thromboprophylaxis, the utility of laboratory‐based tests that assess aspirin efficacy have not been evaluated. We sought to determine the relationship between platelet aggregation testing and aspirin dose adjustment on thrombosis rates in this population. Methods: Pediatric patients undergoing cardiac surgery who received aspirin and underwent platelet testing were studied retrospectively. Patients were excluded if they were treated with multiple agents or experienced thrombosis before the initiation of aspirin. Thrombosis events within 30 days after initiation of aspirin were recorded. Associations between aspirin responsiveness and thrombosis rate and aspirin dose adjustment and thrombosis rate were assessed with the use of multivariable logistic regression analysis. Results: Suboptimal platelet response to aspirin was detected in 64 of 430 patients (15%) and thrombosis was detected in 11 patients (2.6%). Lack of aspirin responsiveness on initial testing was a significant risk factor for thrombosis (P < .001) independent of age, weight, diagnosis, and initial aspirin dose. Dose escalation based on aspirin testing was performed in 40 of 64 patients, and significantly lower rate of thrombosis was observed in patients who underwent dose escalation compared with those without dose escalation (0/40 vs 9/24, P < .001). By multivariable analysis, the only significant independent risk factor for thrombosis was failure to increase aspirin dose after initial unresponsiveness (P < .001). Conclusions: Current practice of weight‐based aspirin dosing may lead to subtherapeutic platelet inhibition in some children. Aspirin unresponsiveness is associated with increased risk of thrombosis after specific pediatric cardiac surgical procedures. Aspirin dose increase in unresponsive patients is associated with reduced risk of thrombosis.

Relationship Between Transfusion of Blood Products and the Incidence of Thrombotic Complications in Neonates and Infants Undergoing Cardiac Surgery

OBJECTIVESnThe authors hypothesized that transfusion of blood products in neonates and infants undergoing high-risk cardiac surgery in the absence of intraoperative coagulation monitoring increases the risk of thrombotic complications.nnnDESIGNnProspective observational study.nnnSETTINGnNeonates and infants undergoing cardiac surgery at a tertiary pediatric center.nnnPARTICIPANTSnNeonates weighing >2.5 kg and infants ≤12 months of age undergoing elective cardiac surgery with cardiopulmonary bypass were included in this prospective observational study.nnnINTERVENTIONnNone.nnnMEASUREMENTS AND RESULTSnDemographic data, surgical characteristics, transfusion data, and coagulation parameters (thromboelastography and thromboelastometry) were collected. Logistic regression analysis was performed to identify potential determinants of postoperative thrombotic complications. Among the 138 neonates and infants included in the study, 12 (9%) developed a postoperative thrombotic complication. Unadjusted logistic regression analysis confirmed that the number and volume of blood products transfused were associated significantly with the increased incidence of thrombotic complication (odds ratio: 2.78, 95% confidence interval: 1.30-5.94, p = 0.008). This association persisted after adjustment for patients age, the need for deep hypothermic cardiac arrest, and bypass time (odds ratio: 2.23, 95% confidence interval: 1.02-4.87, p = 0.044). The number of blood products transfused was associated with a significant increase in parameters of clot amplitudes measured at cardiac intensive care unit admission, while no difference was reported when measured after the administration of protamine.nnnCONCLUSIONSnThis prospective observational study reports a significant association between transfusion of blood products in neonates and young infants undergoing cardiac surgery and an increased incidence of thrombotic complications in the absence of intraoperative coagulation monitoring.

Elevated preoperative von Willebrand Factor is associated with perioperative thrombosis in infants and neonates with congenital heart disease

Essentials Perioperative thrombosis is a major cause of morbidity and mortality in congenital heart disease. Neonates and infants undergoing repair of congenital heart lesions were prospectively followed. Elevated von Willebrand factor (VWF) to ADAMTS‐13 activity ratios typified the postoperative period. Thrombosis was associated with preoperative VWF activity and cryoprecipitate transfusion