SUNDAY AFTERNOON, APRIL 25, 2004

3:00 p.m.

C. WALTON LILLEHEI RESIDENT FORUM SESSION

North Bldg., Rm 204, Metro Toronto Convention Centre

(7 minutes presentation, 8 minutes discussion)

Moderators: Robert C. Robbins

Ross M. Ungerleider

L1. The Sequential Contraction of the Myocardial Band

Manuel Castella*, Gerald D. Buckberg, Saleh Saleh*, Mory Gharib*; Los Angeles, CA, Pasadena, CA

OBJECTIVE: Analyse sequential contraction pathway along the myocardial band of Ton-rent Guasp's "rope-heart model" to determine if structure/function relationship exists in the outer shell wrapping around both ventricles, and inner helical apical loop containing reciprocal descending and ascending spiral segments.

METHODS: In 24 pigs, temporal contraction by sonomicrometer crystals ECG, and Millar pressure transducers followed dP/dt and intraventricular pressure. We traced trajectory contractile patterns, evaluated active suction for ventricular filling, anisotropic function, and Purkinje activation/muscular contraction complexes.

RESULTS: Contractile sequence proceeded from right to left in basal loop, down the descending and up the ascending apical loop segments. The epicardial basal loop stiff outer shell, always contracted before endocardium of the underlying helix. Crystal site placement defined contractile trajectory as transverse in basal vs. oblique in apical loop, subendocardial in descending and subepicardial in ascending segments. Mean shortening fraction averaged 18±3%, with an anisotropic deformation allowing 5±1% more apical than basal contraction. The outer ascending segment followed inner descending contraction by 82±23 msec, and finishing 80±20 msec later. Consequently, isovolumetric relaxation was absent, and active contraction caused suction for venous return; These sequential helical time intervals were shortened by dopamine (~39±5), and lengthened by esmolol (~103±30 msec.) to accentuate and diminish contraction and suction.

CONCLUSIONS: Contractile sequence followed the rope like myocardial band model to contradict traditional thinking with a) epicardial before endocardial contraction, despite early endocardial activation, and b) active contraction causes suction for venous return, as passive recoil from isovolumetric relaxation is absent.

*By Invitation

L2. Alterations of Transmural Strains in the Ischemic Border Zone During Acute Mid-Circumflex Occlusion

Filiberto Rodriguez*, Frank Longer*, Katherine B Harrington*, Allen Cheng*, George T. Daughters*, John C. Criscione*, Neil B. Ingels, Jr.*, D. Craig Miller; Stanford and Palo Alto, CA, College Station, TX

OBJECTIVE: The left ventricle consists of helically oriented myofibers connected by a collagen weave to form transmural laminar "sheets". Normal LV wall thickening mechanics are complex with 15% fiber shortening resulting in 40% radial wall thickening and 60% ejection fraction via laminar shear, extension and thinning. Regional ischemia should alter such LV mechanics, and the ischemic "border zone" may be important in the progression of ischemic cardiomyopathy. For better mechanistic understanding, we examined cardiac wall deformation and microstructure in the ischemic border zone.

METHODS: Nine sheep had radiopaque LV markers implanted to measure fractional area shrinkage [FAS = 100*(regional area_max - regional area_min)/regional area_max)]; 3 transmural bead columns were implanted at the equatorial level in the mid-lateral wall. 3D Marker coordinates were obtained with biplane video fluoroscopy. Data were acquired before and during 70 seconds of mid-circumflex occlusion (distal to first obtuse marginal). Myocardial deformations were quantified at 20% (subepicardial), 50% (mid), and 80% (subendocardial) wall depths using strain analysis of 3D bead displacement from end-diastole to mid-ejection along circumferential (X1), longitudinal (X2), and radial (X3) axes. Strains were transformed into fiber (Xf) and sheet (Xs) coordinates in 5 sheep using quantitative histological measurements of transmural myofibrillar and myolaminar angles.

RESULTS: Ischemia caused significant hemodynamic insult and decreased posterolateral and posteroseptal FAS (Table). FAS revealed that the transmural bead set was in the ischemic border between the posterolateral and anterolateral territories. Interestingly, FAS increased in the remote anteroseptal region. In the ischemic border, subepicardial circumferential (E11)shortening changed to lengthening, circumferential-radial shear (E13)increased, fiber-sheet shear (Efs)reversed, and midwall Efs increased.

Data expressed as Mean±SD. p-values from t-test for pairedobservations.

CONCLUSIONS: Increased regional FAS in non-ischemic sites reflects unloading of remote myocardium. Such unloading reverses E11and Efsand increases E13in the subepicardium while increasing midwall Efsshear in the border zone. These changes in transmural shears likely reflect "slipping" along ischemic border cleavage planes; such dyskinetic wall motion causes increased stretch and stress, which is myopathic. Understanding these processes is important for rational development of surgical therapies for ischemic cardiomyopathy.

*By Invitation

L3. Combined Endothelial and Myocardial Protection by Endothelin Antagonism Enhances Transplant Allograft Preservation

Paul W. M. Fedak*, Vivek Rao, Danny Ramzy*, Subodh Verma*, Laura Tumiati*, Patty Boylen*, Santiago Miriuka*, Richard D. Weisel; Toronto, ON, Canada

OBJECTIVE: Endothelin (ET-1) is a potent inflammatory peptide associated with myocardial dysfunction, coronary vasculopathy, and reduced survival after cardiac transplantation. We hypothesized that ET-1 antagonism during cardiac allograft storage would limit early endothelial dysfunction and improve myocardial performance following transplantation.

METHODS: Orthotropic transplants were performed in Yorkshire pigs (70kg) after cardioplegic arrest and a 6-hour period of ischemic storage. Treatment during storage with intermittent donor blood perfusion (CONTROL, n=8) was compared to ET-1 antagonist (ETA)-enhanced donor blood perfusion (TREATMENT, n=8) using l00μM Bosentan. A macrovascular tissue bath apparatus determined coronary endothelial function. LV performance was assessed by pressure-volume loop analysis after caval occlusion using a Millar micromanometer and conductance catheter to determine preload recruitable stroke work (PRSW). Myocardial ET-1 protein expression was measured by ELISA; TNFa and TGFβ expression by immunoblotting. Oxidative stress was inferred by 8-isoprostane levels. Myocardial metabolism was assessed by measuring the extraction or production of oxygen, acid and lactate by the heart.

RESULTS: Endothelial-dependent coronary vasoreactivity (response to bradykinin) was diminished from baseline in transplanted hearts at 48 hrs after transplantation, but not earlier. Endothelial-independent coronary vasoreactivity (response to sodium nitroprusside) was unchanged in these hearts confirming an underlying endothelial-specific coronary vasomotor dysfunction. Notably, ETA exposure during preservation significantly limited coronary endothelial dysfunction 48 hrs after reperfusion (%Emax to bradykinin: 67±6 v 45±2%, P=0.001). In addition, weaning from CPB (7/8 v 5/8) and LV performance after transplantation was greater in ETA treated hearts (PRSW as % of baseline: 88±6 v 46±2%, P=0.02). Myocardial ET-1 expression increased during reperfusion following transplantation (36±8 v 15±4 fmol/mg, P=0.001) and the rise was comparable in both groups. TNFa was decreased with ETA treatment (109±13 v 133±14 units, P=0.02)while TGFβ did not change (P=0.86). Isoprostane, oxygen, acid, and lactate levels were similar between groups excluding oxidative stress and enhanced metabolic recovery as the underlying mechanism of benefit.

CONCLUSIONS: These data indicate that ET-1 accumulates in cardiac allografts during storage and directly contributes to early endothelial and myocardial dysfunction after transplantation. ETA-enhanced donor blood perfusion during allograft preservation is a clinically applicable procedure that limits endothelial injury and enhances ventricular recovery after transplantation.

*By Invitation

L4. Adenovirus-Mediated Modulation of Phosphatidylinositol 3-Kinase Signaling Reduces Intimal Hyperplasia in Aortocoronary Saphenous Vein Grafts

Jonathan A. Hata*, Jason A. Petrofski*, Jianhua Huang*, Jacob N Schroder*, Matthew L. Williams*, Michael T. Corwin*, Andre M. Jakoi*, Thomas R. Gehrig*, Christopher D. Kontos*, Carmelo A. Milano*; Durham, NC

OBJECTIVE: Approximately 50% of human saphenous vein grafts (SVGs) are occluded 10 years after coronary artery bypass grafting (CABC). Intimal hyperplasia (IH) is an initial step in SVG occlusion and is marked by vascular smooth muscle cell (VSMC) proliferation. The enzyme phosphatidylinositol (PI) 3-kinase and its downstream regulator, the inositol 3-phosphatase PTEN, are key regulators of IH. Studies demonstrate that PTEN overexpression in VSMCs inhibits their proliferation, migration, and survival. This study investigates whether treatment of SVGs with an adenoviral vector encoding the PTEN transgene (AdPTEN) can limit SVG IH in a large-animal CABG model.

METHODS: Twenty-three dogs (30 kg) underwent CABG to the left anterior descending artery using autologous reversed saphenous vein. SVGs were treated with saline (CON, n=9), empty adenovirus (AdEV, n=7), or AdPTEN (n=7). The replication-deficient adenoviruses (5x10¹¹ particles in 1 mL saline) were delivered by distending the SVG to 10 mmHg with adenovirus-containing solution for 20 minutes prior to anastomosis. Following AdPTEN infection, segments of each SVG were cultured ex vivo for 48 hrs to allow transgene expression, then homogenized and PTEN overexpression confirmed by Western blotting. In addition, a subset of dogs received SVGs treated with a marker transgene (Ad_gal, n=3) and were sacrificed on post-operative day (POD) 3 to confirm the distribution of transgene expression. Arteriograms performed on POD 30 and 90 assessed SVG patency. At POD 90, dogs were sacrificed and SVGs histologically analyzed to quantify IH. Data are expressed as mean±SEM; statistical analysis was performed across groups using ANOVA and between groups using Student's t-test.

RESULTS: In Ad_gal-infected SVGs, transgene expression was diffusely distributed throughout the intima, demonstrating efficient transgene delivery. Western blotting revealed marked PTEN overexpression in vessel segments infected with AdPTEN compared to control vessels. Arteriograms on POD 30 and 90 revealed all SVGs to be patent. The intima/media ratio was significantly lower in AdPTEN-treated SVGs compared to both AdEV and CON (0.50±0.05 vs 1.37±0.2 and 1.11±0.14; p<0.005). In addition, AdPTEN SVGs demonstrated reduced total intimal area compared to AdEV and CON (1.39±0.13 vs 2.28±0.37 and 2.57±0.4, mm²; p<0.05). Medial area and maximum/minimum wall thickness were not significantly different among groups.

CONCLUSIONS: This study demonstrates that adenovirus-mediated expression of PTEN inhibits aortocoronary SVG IH in a clinically relevant, large-animal model. These results suggest that modulation of the PI 3-kinase pathway via PTEN overexpression may represent a novel potential therapy to prevent IH after CABG.

*By Invitation

L5. Surgical Treatment for Congestive Heart Failure Using Autologous Adult Stem Cell Transplantation: A Prospective Randomized Study

Amit N. Patel*, Roberto F. Vina*, Luis Geffner*, Robert Kormos, Harold C. Urschel, Jr., Federico Benetti*; Pittsburgh, PA, Rosario, Argentina, Dallas, TX

OBJECTIVE: Autologous adult stem cell transplantation has been used to treat many diseases. The use in cardiovascular disease has only recently been performed. The human experience with a novel epicardial technique to deploy stem cells was compared to conventional therapy.

METHODS: After 1RB and government approval, adult autologous stem cell transplantation (CD34+/CD45_) was performed in patients with coronary artery disease and an ejection fraction of <35% who are going to have primary OPCAB. Preoperatively the patients had an echocardiogram, stress thallium imaging SPECT, and a cardiac catheterization. These imaging modalities were used in identifying ischemic regions of heart and to guide in mapping for injection of the stem cells. The patients were prospectively randomized before operative therapy was performed. Patient follow-up was one, three, and six months with echocardiogram, SPECT, and angiography.

RESULTS: There were twenty patients enrolled in the study. In the study group, ten patients had successful transplantation of autologous stem cells into ischemic myocardium. The other ten patients in the control group only had OPCAB. There were 8 males and 2 females in each group. The median number of grafts performed was 1 in both groups. On angiographic follow-up, all grafts were patent at 6 months. At 1, 3 and 6 months, there was improvement in perfusion on SPECT imaging in areas injected with stem cells. There were no perioperative arrhythmias, neurological or ischemic myocardial events in either group.

CONCLUSIONS: Improvement in cardiac function after autologous stem cell transplantation is promising. Further investigation is required to quantify the cellular effects of the therapy.

*By Invitation

L6. Targeted Overexpression of Leukemia Inhibitory Factor Preserves Myocardium in Postinfarction Heart Failure

Mark F. Berry*, Timothy J. Pirolli*, Vasant Jayasankar*, Kevin J. Morine*, Mirielle A. Moise*, Omar Fisher*, Timothy J. Gardner, Paul H. Patterson*, Y. Joseph Woo; Philadelphia, PA, Pasadena, CA

OBJECTIVE: Ischemic cardiomyopathy is an increasingly prevalent condition with significant medical and economic implications. Leukemia inhibitory factor (LIF) is a cytokine that regulates the growth, differentiation, and function of many embryonic and adult tissues, including the heart. This study examined the effects of viral gene transfer of LIF in infarcted rat hearts.

METHODS: Lewis rats underwent ligation of the left anterior descending coronary artery and direct intramyocardial injection of replication-deficient recombinant adenovirus encoding LIF (n=6) or null virus as control (n=6) into the area of acute ischemia and the bordering myocardium. After six weeks, the following was evaluated: left ventricular (LV) geometry and architecture by histology; myocardial fibrosis by Masson's Trichrome staining; and cardiac function by in vivo pressure-volume conductance catheter measurements.

RESULTS: Rats treated with Adeno-LIF had more preserved myocardium in both the infarct and borderzone, with less fibrosis in the infarct region (LIF 27.5±3.7% fibrosis of infarct region vs Control 40.0±3.2%, p<0.05). LIF treated animals had improved thickness of the borderzone myocardium (1.9±0.1 mm vs 1.5±0.1 mm, p<0.05) with less dilation of the LV cavity (LIF LV cavity diameter 9.2±0.2 mm vs Control 9.9±0.2 mm, p<0.05). LIF treated animals had improved cardiac contractility, indicated by an upward and leftward shift in the pressure-volume relationships of their hearts compared to control hearts (LIF slope of maximum change in pressure over time versus end diastolic volume relationship 61.4±5.0 mm Hg/sec/µL vs Control 39.8±5.3 mm Hg/sec/µL, p<0.05).

CONCLUSIONS: Adenoviral-mediated myocardial gene transfer of LIF results in preservation of cardiac tissue, geometry, and contractile function 6 weeks after myocardial infarction in rats. Treatment with LIF ultimately may be useful in preventing the development of ischemic cardiomyopathy.

*By Invitation

L7. The Evolution of Ischemic Spinal Cord Injury in Inflammation, Function, and Cytoarchitecture and the Effects of Adenosine A2A Receptor Activation

T. Brett Reece*, David 0. Okonkwo*, Peter I. Ellman*, Patrick S. Warren*, Irving L. Kron, Curtis G. Tribble, John A. Kern*; Charlottesville, VA

OBJECTIVE: The progression of ischemic spinal cord injury following reperfusion has not been defined in terms of the time course of changes in function, Cytoarchitecture and inflammatory markers. The aim of this study is to document progression of this injury and demonstrate that adenosine A2A receptor activation with ATL-146e limits detrimental changes in each of these aspects.

METHODS: Mature swine underwent 30min of descending aortic occlusion. They were divided into 3 groups (Sham thoracotomy, IR: 30min ischemia, and ATL: 30min ischemia + ATL-146e for the first 3h reperfusion) .Subgroups(each n=8) were sacrificed at Oh, 3h, 6h, 12h, 24h, and 48h of reperfusion. Functional outcomes were followed in the 48h groups. The spinal cord tissue was evaluated for neuronal viability, microtubule associated protein-2 preservation and neutrophil sequestration (myeloperoxidase assay, MPO). Finally, neuronal tissue, CSF and serum were evaluated for TNF-a using an ELISA kit.

RESULTS: Function was significantly impaired at 24h, 36h, and 48h in IR compared to both Sham and ATL (all p<0.05, e.g. 48h 2.0±0.5 vs 5.0±0 and 4.5±0.3). Neuronal Viability and MAP-2 staining by percentage of gray matter was significantly preserved in both Sham and ATL compared to control at both 24h and 48h (p<0.05). The spinal cord tissue MPO levels were significantly higher in IR than Sham and ATL at both 24h and 48h. The TNF levels (pg/ml) in serum and CSF were low in all groups, but the spinal cord levels were significantly higher in IR compared to Sham and ATL at 6h (187±75 vs 4±2.6 and 9±2.4) and 12h (184±55 vs 4±1.9 and 5±2.3)(all p<0.05).

CONCLUSIONS: Spinal cord ischemia leads to significant changes in neutrophil sequestration, MAP-2 staining and neuronal viability by H&E within 24 hours of reperfusion. Most importantly, despite low serum and CSF levels of TNF-α, spinal cord parenchymal levels of TNF-α rise significantly by 6-12 hours of reperfusion. Adenosine A_2A receptor activation prevents the rise in cytokine levels and markers of cellular inflammation, which may be critical in the preservation of neuronal function and Cytoarchitecture following ischemia/reperfusion.

*By Invitation

L8. Gene Transfer of Soluble TIE2 Ameliorates Pulmonary Hypertension in Rodents

Masakuni Kido*, Lingling Du*, Stuart W. Jamieson, Patricia A. Thistlethwaite*; San Diego, CA

OBJECTIVE: Overexpression of Angiopoietin-1 (Ang-1) in the lung has been associated with different forms of human pulmonary hypertension. We hypothesized that inhibiting the Ang-1 signaling pathway in the lung, by administration of a competitive inhibitor which blocks Ang-1 binding to its receptor, TIE2, would block the development of pulmonary hypertensive vasculopathy in a rodent model.

METHODS: Two rodent models of pulmonary hypertension were tested: 1) animals with pulmonary hypertension induced by constitutive Ang-1 expression in pulmonary vascular smooth muscle, and 2) animals with pulmonary hypertension induced by administration of monocrotaline. We injected 2x10'° genomic particles of an adenoassociated virus containing an extracellular fragment of the TIE2 receptor (AAV-sTIE2) into main pulmonary artery of 30 rats with either Ang-1 or monocrotaline-induced pulmonary hypertension, while using adenoassociated virus-lacZ (AAV-lacZ) and carrier-injected rats as controls. All animals underwent survival surgery and were sacrificed at serial timepoints post gene delivery. At each timepoint, pulmonary artery pressures were measured and pulmonary angiography performed. Lungs were harvested for pathologic analysis, mRNA and protein analysis, and in situ hybridization to localize gene expression.

RESULTS: Pulmonary artery pressures of rats overexpressing Ang-1 in the lung and rats treated with monocrotaline were significantly increased compared to control groups (p<0.01) at all timepoints. Administration of AAV-sTIE2 ameliorated pulmonary hypertension in both groups (from 36±2.4mmHg to 18±1.6mmHg in the Ang-1 group, p<0.01; from 45±2.3mmHg to 17±1.3mmHg in the monocrotaline group, p<0.01). Pathologic analysis of lungs treated with AAV-sTIE2 demonstrated reversal of smooth muscle cell proliferation within the medial layer of arterioles. Pulmonary angiography confirmed reversal of small pulmonary vessel occlusion in animals treated with AAV-sTIE2.

CONCLUSIONS: Molecular blocking of the interaction between Ang-1 and its endodielial receptor, TIE2, in the lung reverses pulmonary hypertension in two animal models of the disease. These experiments suggest a new strategy for treating pulmonary hypertension, based on the molecular biology of the pulmonary vascular wall.

5:00 p.m. ADJOURN TO WELCOME RECEPTION EXHIBIT HALL

*By Invitation