Increased vascular endothelial growth factor in patients with cyanotic congenital heart disease may not be normalized after fontan type operation

408A

JACC

ABSTRACTS - Pediatric Cardiology 12:48 p.m.

1121 MP-125

Evaluation of the Accuracy of a New Semiautomatic 3-D Program (PMAP) by Quantifying Right Ventrlcular Volume Using RseI-Tlme 3-D Imaging: Studies in an In Vitro Model Duplicating Human RV Anatomy

Lisetfe S.L. Oei. Sebastian T. Schindera, Xiaokui Li, Petre S. Mehwald, Johnny Kuo, John Castellucci, Olaf T. von Ramm, Deniz Kececioglu, David J. Sahn, Oregon Hea/th& Science University, Portland, Oregon, Universitaetsklinik, Freiburg. Germany,

Background:We assessed the accuracy of a custom semiautomated 3D analysis program (PMAP, Duke Center for Emerging Cardiovascular Technologies) for measuring dght ventdcular volumes (RW) and stroke volumes (SV) acquired from real time 3D (RT3D) imaging of static and pulsatile RV cavity models. Methods: We used RT3D (Volumstdcs Imaging System) to image 10 static and 5 dynamic elastic RV latex shells cast from human hearts in a pulsatile model where actual volume was determined by water displacement. Offline measurements were made by manually tracing (Volumetrics program) a family of 810 dedved B-scans across the imaged volume, or the new PMAP program which semiautomatically boundary detects 32 deformed circular profiles between the apex, base and outflow tract (32 radii [r] emanating from the center of each circle) using all the 3D data guided by only 2 or 3 selected traced B-scans. Volume was calculated according to the formula, ~*B*(r2)*(1/32)*h, where h is height of one slice in the stack of circles. Results: There was a strong correlation between the static RVV results of the PMAP pmgrem with the true volume. PMAP tended to slightly overestimate the outcome (percent difference: 0.91 ± 8.71%, r = 0.94) compared to the manual Volumetrics Imaging program (percent difference: 0.60 ± 6.91%, r = 0.97). For the dynamic stroke volumes (SV) from the human casts, PMAP (percent difference: -4.43 ± 13.98%) versus the Volumetrics manuel tracing program (percent difference: -7.90 ± 14.21%) results were similar.

I\

March 6, 2002 1:12 p.m.

1121 MP-127

:J Spatio-Temporal Descri~ion of the Development of the Mouse Cardiac Conduction System Using a cGATA-6 Gene Enhancer Marker

Amy L. Juraszek, Angela V. Edwards, Aimee Phelps, John B. Burch, Andy Wsssels, Medical University of South Carolina, Charleston, South Carolina, Fox Chase Cancer Center, Philadephia, Pennsylvania.

Background: A fully developed cardiac conduction system is essential for coordinated cardiac contraction and unidirectional blood flow through the head. Recently, a cGATA-6/ lacZ transgenic mouse model has been generated in which the transgene expression is localized to the proximal region of the atdoventricuisr (AV) conduction system {i.e. AV node) and the right AV ring bundle (Davis et at., Mech. Dev., Oct 2001) in the postnatal heart. Methods: [-1.5/+0.8]cGATA-6/lacZ 2.3 kb mouse embryos were obtained at embryonic day (ED) 7.5 to ED18. The specimens were stained for lecZ, paraffin embedded, serially sectioned, counterstained with nuclear fast red and examined by light microscopy. Digital images of the sections were then used to generate three dimensional reconstructions using 3D-DOCTOR software (Able Software). ResultS: At the paired heart field stage (ED7.5), transgene expression is localized to a subpopulation of cells in the lateral portions of the precardiac mesoderm. At ED8.5 the expression is found at the AV junction in the myocardium adjacent to the developing endocardist cushions. From the AV junction two lacZ positive bends extend towards the apex over the anterior and posterior surfaces of the left ventricle. From ED9.5 onward the ventricular transgene expression disappears and the lacZ positive region becomes more restricted to the right AV junction including the region of the developing AV node. As the atrial and ventricuiar myocardium become separated by the fusion of sulcus and cushion derived tissues (ED14 and beyond), the transgene expression becomes incorporated into the right atrial myocardium along the lateral and posterior rim of the tricuspid valve annutus and in the area of the developing AV node. Conclusion: The cGATA-6/lacZ transgenic construct studied identifies a distinct subpopulation of cells in the precardiac mesodarm at eady stages of development. At advanced stages of development the cGATA-6/lacZ expression is found in the proximal portion of the AV conduction system and dght AV ring bundle. This cGATA6 construct appears to be the earliest reported marker for the developing conduction system. Further cell fate studies using a cre°lox approach are in progress. 1:24 p,m. 1121 MP-128

Shortened O u t f l o w Tract Leads to Altered Cardiac

Looping After Neural Crest Ablation Taiat Mesud Yel~uz. Karen L. Waldo, Donna H. Kumiski, Harriett A. Stadt, Raymond R. Wolfe, Linda Leatherbury, Margaret L. Kirby, Duke University Medical Center, Durham, North Carolina.

1:00 p.m. 1121MP-126

Molecular C l o n i n g and Characterization of a Potential

Candidate Gene for Nonsyndromic Atrioventricular Septal Defect o n 11)31-1p21 Yopn Zhao, Xianmin Meng, Huiqing Can, Liguo Mi, Yingjie Wei, Choong-Chin Liew, Jinfeng Ding, Cardiovascular Institute & Fu Wai Heart Hospital, PUMC & CAMS, Beijing, People's Republic of China, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts. Background: Aifhough atrioventdcular septal defect(AVSD) is commonly associated with Down syndrome, its isolated form is not linked to the Down syndrome critical region. Genetic studies suggest that AVSD is a genetically heterogeneous disorder and its isolated form differs from syndromic one. Recent efforts have demonstrated that one genetic locus for nonsyndromic AVSD maps to chromosome lp31-tp21. While exa~y which gene(s) account for nonsyndromic AVSD remains poody understood. The aim of this study was to screen for the potential candidate genes for involvement in nonsyndromic AVSD. Methods: We applied a bioinformatics-besed approach to analyze the genes located on 1p31-1p21 against the Genome Database, and their expression level and tissue specificity were estimated by the number of expressed sequence tags that were included into the UniGene cluster. Biochemical studies were employed for further confirmation. Results: Of some dozens of genes, we identified a novel kinase gana located on 1p31 and named it p93 because the 835 amino acid gene product has a predicted Mr of 93 kDa(GenBank acc. AFl16826). Sequence analysis demonstrated that p93 may be a distant MAPKKKs member of integdn-linked kinase(ILK) family. Subsequently Northern blot and 76tissue array analysis(Clontech) showed: 1) p93 was expressed in heart, but undetectable in any other tissues. 2) Right atdoventdcular p93 mRNA level was higher than the counterpart of left. 3) p93 was significantly highly expressed in intarventricular septum and apex of heart. Immunohistochemistry revealed that p93 was more highly expressed in the nucleus of embryonic cardiac myocytes than in adult. Of note, p93 substantially decreased in myocardiums of subjects with tetralogy of Fallot comparison with normal ones. In vitro kinase analysis showed p93 could autophosphorylate while its dominant negative form(K490R) couldn't, which indicated that p93 is a functional kinase. Conclusions: p93 is a cardiac-specific and developmentally-regulated kinase, and it may be a strong candidate gene involving in nonsyndromic AVSD.

Background - Congenital conotruncal malformations are associated with the highest mortality dsk in utero and after postnatal surgical repair, and frequently involve dextroposed aorta. The pathogenesis of daxtroposed aorta is not known but is thought to be due to abnormal looping and/or wedging of the outflow tract during early heart development. The elongation of the outflow limb of the cardiac tube is a critical step in the normal pro. cess of looping insofar as it provides required extra material to lengthen the heart tube. We examined cardiac morphology in an experimental model of dextroposad aorta to determine whether altered lengthening of the outflow limb plays a role in dextroposition of the aorta. Methods and Results - Hearts were examined in normal chick embryos and compared with those in cardiac neural crest-ablated embryos using time-lapse vldsophotography, scanning electron microscopy and histological sectioning (collected embryos: n=118; analyzed embryos: n=88). We found that in neural crest-ablated embryos the inflow and outflow limbs of the looped cardiac tube were malpositioned with respect to each other, the inner curvature of the looped tube was diminished, and the outflow limb straighter and displaced cranially in a manner consistent with diminished length. Furthermore, we found that movement of cells from the secondary heart field into the myocardium of the outflow tract was significantly less as shown by a paucity of HNK-1 positive cells in the secondary heart field and outflow myocardium. Conclusions'- The data are consistent with previous experiments from many labs showing that normal ldoping and wedging are essential for normal alignment of the aorta with the left ventricle. Our results show that addition of myocerdium to the outflow tract is necessary for normal looping and wedging to occur at all, and indicates for the first time that the shortened outflow limb is a result of failure of the outflow tract to lengthen by addition of myocardial cells from the secondary heart field. 1:36 p.m. 1121MP-129

Increased Vascular Endcthellal Growth Factor In Patients With Cyanotic Congenital Heart Disease May Not be Normalized After Fontan l~/pe Operation

K~nii Soda. Masahiko Matsumura, Setsuko Miyenishi, Temi Hospital, Tenrl, Japan, Tenrl Medical Research Institute, Tenrl, Japan. BACKGROUND: There is no data available concerning the change of vascular endothelial growth factor (VEGF) in patients with cyanotic congenital heart disease (C-CHD). PURPOSE: To determine the change of serum concentration of VEGF In patients with CCHD. METHODS: Subjects were divided into 2 groups; A(age 1 - 20 years) and B {age > 20 years). Group A was subdivided into 4 groups; AI, 23 patients with uncorrected CCHD; A2, 13 controls; A3, 8 patients who had C-CHD and were treated with bi-ventdcular

JACC

ABSTRACTS - Pediatric Cardiology 409A

March 6, 2002

repair; A4, 14 patients who had single ventricle and were treated with Fontan type operation. Group B was subdivided into 3 groups; B1, 6 patients with C-CHD palliated by classic Glenn procedure; B2, 6 patients with C-CHD palliated by shunt procedure; B3, 9 healthy controls. There was no significant difference in age among subgroups of A and among subgroups of B. Blood samples were obtained from upper arm veins and serum VEGF was determined by ELISA method. We determined correlation between patient's arterial saturation and serum VEGF in combined group of A1, A2, B1, B2 and B3 and compared level of serum VEGF among groups. Data were expressed as median and inter-quartile range in parenthesis. RESULTS: Serum VEGF was significantly negatively correlated with arterial saturation (r=.48, p<.001). Serum VEGF in A1 was significantly higher than those in A2 and A3 [373(269) pg/ml in A1 vs. 196(121) pg/ml in A2 and 186(87) pg/ml in A3, respectively[, but not higher than VEGF in A4 [257(293) pg,'ml]. Although it did not reach significant level, serum VEGF in A4 tended to be higher than A3 (p=.07). Serum VEGF in B1 and B2 was significantly higher than in B3 [269(379) pg/ml in B1 and 657(382) pg/ml in B2 vs. 225(105) pg/ml in B3, respectively]. There was no significant difference in serum VEGF level between B1 and B2. CONCLUSIONS: Patients with C-CHD seem to have increased serum VEGF in parallel with the degree of cyanosis. Patients with C-CHD palliated by classic Glenn procedure do have increased serum VEGF, but not more than those palliated by shunt procedure. With bi-ventricular repair, cyanosis disappears and serum VEGF may be normalized. In contrast, with Fontan type operation, cyanosis disappears but serum VEGF may not be normalized 1:48 p.m. 1121MP-130

Leukocyte Adhesion Factor Mac-1 and Migration Inhibitory Factor-Related Protein (MRP) on Granulocyte Plays the Essential Role for Causing Vasculitis in Kawaseki Disease and the Gamma Globulin Therapy Inhibit Leukocyte-Endothelial Cell Adhesion

Rvuii Fukazawa. Yohoko Uchikoba, Yukio Kuramochi, Ei Iksgami, Mitsuhire Kamisago, Yasuhiro Katsube, Takashi Seki, Shunichi Ogawa, Nippon Medical School, Tokyo, Japan.

(Background) We have revealed massive expression of Ca binding protein migration inhibitory factor-related protein(MRP) on circulating granulocyte in acute phase of Kawesaki disease. Newton et. aL (J Immunol 1998 160:1427) reported MRP reinforce the ability of adhesion molecule Mac-l, which suggest the relationship between MRP/ Mac-1 and vasculitis. We quantified leukocytes Mac-1 expression in Kewaseki disease, and evaluated the adhesion ability between cultured human coronary artery endothelial cell and Kawasaki disease patient's peripheral leukocyte. Furthermore, we evaluate the leukocyte-endothelial cell adhesive ability using the patients' plasma, before and after gamma globulin therapy, to see the gamma globulin effect for this system. (Materials and Methods) mRNA was extracted from the Kawasaki disease patients' leukocyte (n=21) and was converted to cDNA by RT-PCR, and Mac-1 expression was evaluated by quantitative PCR (Applied Biosystems; GeneAmp 5700). Patients' leukocyte, labeled with BCECF-AM, exposed to cultured human coronary artery endothelial cell, and leukocyte adhesion assay was performed. Leukocyte adhesion assay was also carded out using the patients' plasma, pre/post gamma globulin therapy. (Result) Mac-I expression was a peak on acute phase of Kawasaki disease and significantly decreased after 1 month of onset. The patients' leukocyte adhesion ability to endothelial cell was significantly increased, which was significantly inhibited by addition of anti-Mac-1 antibody. And the patients' plasma before gamma globulin therapy significantly increased leukocyteendothelial cell adhesion, which was abolished by the plasma of post gamma globulin therapy. We postulated Mac-1 play the key rote for leukocyte invasion into endothelium, which is the initial step for causing vasculitis. And the gamma globulin therapy is effective through inhibiting leukocyte-endothelial cell adhesion.

1142

POSTER SESSION Pediatric Electrophysiology and Intervention

Monday, March 18, 2002, 3:00 p.m.-5:00 p.m. Georgia World Congress Center, Hall G Presentation Hour: 4:00 p.m,-5:00 p.m. 1142-97

Termination of Intraatrial Reentrant Tachycardia Using Implanted Pacemakers in Patients Following Atrial S w i t c h Repair o f Transposition of the Great Arteries

Gerald A. Sarwer. David J. Bradley, Peter S. Fischbach, Kristen A. George, Sarah S. Leroy, Macdonald Dick, II, University of Michigan Congenital Heart Center, Ann Arbor, Michigan.

Background: Intra-atriat reentrant techycardia (IART) has been associated with patients (pts) following the atrial switch operation (AtrS) for d-transposition of the great arteries who have sick sinus syndrome (SSS). Anti-tachycardic pacing (ATP) has been used to convert IART in these pts but its efficacy using implanted pacemakers and safety have been questioned. Methods: Clinical records of pts post AtrS who had undergone atrial or dual chamber pacemaker placement for SSS were retrospoctively reviewed. Implant details, number and results of ATP attempts, medications, and outcomes were recorded. Results: Of the 29 pts identified, atrial electrode placement was endocardial in 25 and epicardial in 4 and was guided only by pacing threshold data with no attempt to induce and convert IART at implant. 21 (72%) had 1 or more episodes of IART and all were taking digoxin, 5 on beta-blockers, and 2 on amiodarone. 4 of 21 had IART prior to pace-

maker implant without recurrence post implant. 17 underwent ATP for conversion of I to 30 episodes per patient (94 total episodes, median 3/patient). 7 pts experiencing 47 episodes had automatic anti-tachycardic pacemakers. ATP therapy consisted of 15 to 40 beats at a cycle length of 50% to 80% of the IART cycle length (median 67%, minimum 150 ms) delivered at an amplitude and pulse width twice the bradycardia pacing settings. ATP cycle length began at 80% of the IART cycle length and was decreased until conversion. All episodes were converted but many required multiple attempts. 8 ATP attempts increased the IART rate but not the ventricular rate. 4 ATP attempts in 2 pts caused atrial fibrillation that reverted spontaneously to sinus rhythm. All pts were hemodynamically stable in IART before and during ATP. Conclusion: Bradycardia pacing can control IART in some pts. ATP by implanted pacemakers is safe and effective in converting IART in pts post AtrS procedure who are not severely hemodynamically compromised by IART avoiding the need for DC cardioversion. IART induction is not required at pacemaker implant. Pacemakers with atrial overdrive capability should be considered in pts post AtrS with SSS as the incidence of IART is high and ATP is highly effective and safe. 1142-98

Comparison of Adenosine-Sensitive and AdenosineResistant Accessory Pathways in Children

David J. Bradlev. Loren M. Madden, Christopher B. Stefanelli, Elizabeth V. Saarel, Jeremy D. Ashes, Gerald A, Serwer, Peter S. Fischbach, Macdonald Dick, II, University of Michigan Congenital Heart Center, Ann Arbor, Michigan.

Background:Adenosine (Adn) is routinely used during electrophysiolgic study (EPS) of patients with supravantricular tachycardia (SVT) to assess presence of accessory atrioventricular pathways lAP) and verify success of radiofrequency ablation (RFA). Adenosine blocks conduction in the atrioventricu)ar node, but generally not in APe. We investigated the incidence and properties of Adn-sensitive APs. Methods:The electrophysiologic patient database at the University of Michigan Congenital Heart Center was queried for all patients with accessory pathways and SVT undergoing EPS between 4-1995 and 4-2001. Clinical records and EPS tracings were reviewed. Patients received Adn during ventricular pacing at a standard intravenous close of 200mcg/kg, with a maximum dose of 12mg was used. All patients were under 21 years of age (median 11.6 years). Results: A total of 151 patients with APe and SVT were identified; Adn was used in 132 patients (137 APe). Of these, 20 APs (15%) blocked with adenosine. RFA was performed on 85% (117/137) APe, and was successful in 94% (15/16) Adn-sensitive, v 92% (93/ 101 ) Adn-insensitive APe (p=ns). Adn-sensitive and -insensitive APe did not differ significantly with regard to patient age (10.6±5.8 v 11.4±4.7 years), gender (47% v 55% boys), or SVT cycle length (314±39 v 315±46ms), procedure times, total RF applications or rate of ablation success. Adn-sensitive and insensitive pathways did not segregate by septal v free-wall (35% v 28% septal, p--0.16), or left v right sided (63% v 60% left, p=0.36) locations. In contrast, Ado-sensitive APe were less likely to demonstrate pre-excitation in sinus rhythm (37% v 61%, p=0.04), and had significantly longer antegrade effective refractory periods (ERP) when pre-excitation was present (340-Z42v 278+58ms; p=0.05). Retrograde ERP also trended longer in Adn-sensitive pathways (270~57 v 259±43) though this difference was not statistically significant (p=0.10). Conclualona:As Adn-sensitive APs occur frequently in pediatric patients, Adn is not an absolute test of AP presence. Adn-sensitive APs are significantly less likely than Adnresistant APs to cause pre-excitation, and have longer entegrade ERPs when they do. 1142-99

B i o l o g i c Response to the HELEX TM Septal Occluder implantation in the Canine Heart

Geoffrey K. Lane. Nicholas P. Macri, John F. Rhodes, C.Igcr Mesia, Lourdes R. Prieto, Paul L. Sawyer, Melanie M. Manning, Dens J. Anderson, Even M. Zahn, Larry A. Latson, The Cleveland Clinic Foundation, Cleveland, Ohio, W. L Gore & Associates Inc, Flagstaff, Arizona.

Background:The HELEXTM device has recently been used in humans for transcathater secundum atrial septal defect (ASD) closure. We report the data on the biologic response to implantation of this device in the canine heart. Methods: Histologic data from 23 animals enrolled in non-randomized prospective studies conducted at W.L. Gore & Associates Inc and The Cleveland Clinic Foundation were analyzed. Animals had either a surgically created or a percutaneously created ASD and immediate implantation of a HELEXTM device, Animals were sacrificed at intervals from 2 days to 1 year. Devices were examined grossly, with multiple sections of the device and adjacent atrial septum assessed for fibrous connective tissue (FCT) and endothelial-like cell coverage. Distal organs were examined for the presence of thrombo-emboli in animals with implanted devices _>30 days, Results: FCT completely covered the left atrial component in 12/13 devices, which had been implanted _>30 days. One device at 6 months post implantation had complete coverage of the right atrial side with extensive coverage of the left atrial side. Endothelial-like cell coverage was muitifocal or greater in 10/13 devices implanted -> 30 days. One device examined by scanning electron microscopy and immunohistochemistry (for factor VIII) confirmed that the endothelial-like cells seen with bight microscopy were true endothelial cells. There was a trend for greater FCT and endothelial cell coverage of devices with time. There was no evidence of distal organ thromboembolism in any animal examined. Conclusions: The biologic response to implantation of the HELEXTM device appears to be progressive with an initial fibrous connective tissue coverage followed by endothetialization. There was complete coverage of the left atrial side of the device with fibrous connective tissue by 30 clays post implantation in almost all cases with no evidence of distal thromboembolism. These data support the notion that the HELEXTM device is biocompatible. Further research into the process of endothelialization of this and other transcatheter devices is needed.