WEDNESDAY MORNING, MAY 3, 2000

7:00 a.m. EMERGING TECHNOLOGIES AND TECHNIQUES

Constitution Hall, Metro Toronto Convention Centre

Moderators: Robert W. Emery, M.D.

Hani Shennib, M.D.

T1. Computer Enhanced "Robotic" Cardiac Surgery - First Clinical Results in 100 Patients

Friedrich W. Mohr, Volkmar Falk*, Anno Diegeler*, Ralf Krakor*, Jan F. Gummert*, Thomas Walther* and Ruediger Autschbach*, Leipzig, Germany

OBJECTIVE: A computer enhanced instrumentation system was tested to enable total endoscopic cardiac surgery.

METHODS: The daVinci™ system provides a 3-D videoscopic image and allows remote, tremorfree and scaled control of endowrist instruments with 6 degrees of freedom. We used the system in 83 patients for CABG and in 17 patients for endoscopic mitral valve repair. The system was used for ITA-take down followed by minithoracotomy (n=44), to perform the coronary anastomoses in standard sternotomy CABG partially in off pump technique (n=15), or total endoscopic coronary artery bypass grafting of the left ITA to the LAD (TECAB, n = 24). In 17 with non-ischemic mitral valve insufficiency the mitral valve was repaired (MVR). Closed-chest cardiopulmonary bypass with cardioplegic arrest (Port-Access technique) was used for TECAB and MVR.

RESULTS: ITA take down was successful in 58/61 pts. in the MIDCAB group and after a steep learning curve is currently performed in less than 50 minutes (44 ± 13 minutes). Endoscopic ITA take-down decreased the size of the mini thoracotomy (6.8 ± 2.3cm). Injury to the ITA with the need of conversion to sternotomy occurred in 2/83 patients (2.4%) TECAB was completed in 19/24 cases. In 5 pts. conversion to an successful open approach was necessary to safely identify the course of LAD in 3 pts. Angiographic control after 3 months revealed a 100% patency (19/19) in the successful TECAB patients and one graft occlusion (1/92 anastomoses) in all other patients. One patient in the CABG group died from a stroke. In the MVR group one patient with Barlow disease had residual MI II and minimal invasive MVR was immediately followed. In the remaining 16/ 17 patients the primary repair was successful.

CONCLUSIONS: The da Vinci™ system allows for precise tissue handling and enables the endoscopic performance of cardiac surgical tasks that require a high degree of dexterity (coronary anastomosis, MVR). No technical mishaps have occurred. The steep learning curve with long procedural times in the beginning may be preventable by the future use of simulation technology. This experimental study may pave the way for fully endoscopic computer guided surgery.

*By Invitation

T2. Animal and Clinical Study of a New Sutureless Anastomotic Device for the Proximal SVG Anastomosis.

Antonio M. Calafiore, Yaron Barel*, Giuseppe Vitolla*, Assaf Dekel*, Chieti, Italy; Haifa and Herzelia, Israel.

OBJECTIVE: Evaluate the safety and efficacy of a new self expanding nitinol Aortic Anastomotic Device (AAD), designed for off-pump proximal SVG anastomosis without manipulation of the aorta.

METHODS and RESULTS: 16 sheep were used as the animal model. 45 SVG anastomosis were created (patency verified by flow meter), 32 using the AAD and 13 using conventional hand suturing techniques. All procedures were done off pump by a cardiovascular specialist. At sacrifice patency rates were 28 of 32 for AAD bypasses (87.5%) and 11 of 13 for hand sutured bypasses (85.0%). Average anastomosis diameter was 4.5mm and 3.5mm for AAD and hand sutured anastomoses respectively. The AAD was used in 11 coronary patients, 7 on pump and 4 off-pump. Flow meter showed all bypasses to be patent, measurement ranging from 9 to 65 ml/min (distal anastomoses:PDA-9, marginal-4, diagonal-1). The AAD procedure took between 1 to 2 minutes. One AAD did not deploy and was replaced by hand suturing. There were no surgical or post-surgical complications with the AAD. 1 patients had early angiography (5 days), that showed perfect patency (elective angiography was scheduled for 3 months).

CONCLUSIONS: The AAD seems to be a reliable and effective device in creating a sutureless fast SVG anastomosis, especially in beating heart surgery. Avoiding manipulation of the aorta will reduce the risk of embolization from side clamping. The self expanding nature of the device creates a large diameter anastomosis.

*By Invitation

T3. Facilitated Coronary Anastomosis Using the Nitinol Sutured-Clip™ Device.

Arthur C. Hill*, Eric Monnet*, Timothy Maroney* and Renu Virmani*, Omaha, Nebraska; Fort Collins, Colorado; Richmond, Virginia; Washington, District of Columbia

OBJECTIVE: The Sutured-Clip™ device, (Coalescent Surgical, Inc.), was designed to facilitate minimally invasive coronary anastomosis by eliminating the need for suture management, knot tying, and surgical assistance. The device employs the superelastic properties of Nitinol and consists of two components: 1.) a needle/suture delivery system, and; 2.) a detachable Nitinol self-closing wire. The device was tested in the bovine OPCAB model to determine its ease of use and chronic anastomotic functional characteristics.

METHODS: The Sutured-Clip device was tested in 14 calves. RITA-to-coronary artery anastomosis was performed on the beating heart using stabilizer via a left thoracotomy. Functional characteristics of each anastomosis produced by the Sutured-Clip device were evaluated using intra-operative angiography (n=14), intra-operative Doppler flowimetry (n=13), angiography at one week (n=4), angiography at 8 weeks (n=8), and histology at one week (n=4) and 8 weeks (n=8).

RESULTS: All calves survived to completion of the study. All Sutured-Clip anastomoses and grafts were patent and without stenosis by Doppler flowimetry, intra-operative angiography, post-operative angiography, and histology. Histological evaluation snowed smooth neointimal resurfacing of the Nitinol device and interposed tissue with no tissue necrosis and minimal inflammation. Duration of coronary occlusion during anastomosis averaged 14.70 (range: 10-21) minutes. Graft flow by Dopper flowimetry averaged 74.41 (range: 22-180) ml/minute. Two calves, intended for extended long term survival, are still alive and showed angiographic patency at 6 and 10 weeks following device implantation.

CONCLUSIONS: The Nitinol Sutured-Clip device produced high quality interrupted anastomoses in this initial bovine OPCAB study. The device facilitates coronary anastomosis by simplifying and decreasing the amount of manipulation and complexity required in minimally invasive CABG procedures. Nitinol technology also has potential in robotic and conventional surgical applications.

*By Invitation

T4. Modified Glenn Connection for Acute Ischemic Right Ventricular Failure Reverses Secondary Left Ventricular Systolic Dysfunction.

Mark H. Danton*, John G. Byrne*, Kathryn Q. Flores*, Jeffrey S. Martin*, Michael Hsin*, Lawrence H. Cohn and Lishan Aklog*^§, Boston, Massachusetts

OBJECTIVE: Acute ischemic RV failure results in serve haemodynamic compromise, we hypothesize: 1 In addition to reducing LV preload, acute RV dilation may directly compromise LV contractility and 2 By unloading the RV, through a modified Glenn anastomosis, this effect will be attentuated.

METHODS: A SVC main PA connection was performed in 8 pigs and isolated RV ischemic failure induced by selective coronary ligation. Simultaneous measurements of ventricular pressure with volume (RV) and segment length (LV) were made.

RESULTS: Ischemia caused acute RV dilation and, independent of preload, LV systolic and diastolic dysfunction. RV unloading significantly reversed the regional LV contractility deficit without improving diastolic parameters.

CONCLUSION: In acute ischemic RV failure with normal pulmonary vascular resistance a modified Glenn shunt preserves LV systolic performance by limiting RV dilation.

Indicies	Baseline	RV ischemia	Glenn circuit
RV EDV (ml)	102	143†	118‡
LV ESPLR mitiHg/mm	52.1	18.6†	31 .2‡
LV PRSW mm Hg	83.0	45.5†	63.6‡
RV ESPVR mmHg/mL	0.82	0.46†	0.46
RVPRSWmmHg	15.2	11. 6†	11.8
LV Tau ms	35.6	58.8†	51.6
LV dP/dt min	790	476†	533
Cardiac output, L/min	4.01	3.1†	3.0

EDV end diastolic volume, ESPV(L)R end systolic pressure volume (length) relationship. PRSW

precruitable stroke work index.

†<0.05 between baseline and ischemia

‡<0.05 between ischemia and Glenn circuit

^§International Traveling Fellow

*By Invitation

^§T5. Device Based Left Ventricular Shape Change Immediately Reduces Regional Areas and Improves Fractional Shortening Indices in a Canine Cardiomyopathy Model

Patrick M. McCarthy, Kiyotaka Fukamachi*, Masami Takagaki*, Guy Armstrong*, James B. Young*, Cyril J. Schweich*, Todd J. Mortier* and Marc R. Raffe*, Cleveland, Ohio; Auckland, New Zealand; Plymouth, Minnesota

OBJECTIVE: To test the acute effect of left ventricular (LV) shape change on regional areas, contractile indices, and hemodynamics in a heart failure model

.

METHODS: Heart failure was induced in 6 healthy dogs implanted with pacemakers by pacing at 230 beats per minute for an average of 23 days. A novel device, the Myocor™ Myosplint™, was surgically implanted in the LV to produce a calculated 20% or 30% decrease in wall stress by changing LV shape. This was accomplished by placing 3 Myosplint devices perpendicular to the LV long axis, creating a symmetric, bUobular LV. Papillary and mitral level diastolic (PD,MD) and systolic (PS,MS)areas were measured by 2D echo prior to, and following tightening at 20% and 30% stress reduction levels. Papillary level (PFAS), mitral level (MFAS) and mean (FAS_avg) fractional area shortening were calculated. Cardiac output (CO), stroke volume (SV), heart rate (HR), and blood pressure (BP) were measured.

RESULTS: Comparing pre and post tightened values, PD, PS,and MS areas were significantly (p<0.05, paired t-test) lower at 20% and 30% stress reduction. MD area was significantly lower at 30% stress reduction. FAS_av and MFAS were significantly higher at 20% and 30% stress reduction. PFAS was significantly higher at 30% stress reduction. HR, SV, CO, and BP remained unchanged.

CONCLUSIONS: Shape change produced by the Myocor Myosplint immediately improved contractile indices and reduced ventricular area measurements with maintenance of stable hemodynamics. The shape change concept has been clinically replicated as evidenced by the results of our first 2 human cases.

	PD (cm2)_	PS (cm2)_	MD (cm2)_	MS (cm2)_	PFAS	MFAS	FASavg_
Pretighten	19.7	15.9	20.8	16.3	0.23	0.22	0.22
20%	17	10.9	18.6	12.9	0.36	0.30	0.33
Pretighten	18.3	14.7	20.4	16	0.24	0.22	0.23
30%	13.8	9	16.1	10.8	0.35	0.33	0.34

^§Authors have a relationship with Myocor

*By Invitation

8:00 a.m. GENERAL THORACIC SURGERY

FORUM SESSION Room 205

Metro Toronto Convention Centre

Moderators: Robert J. Keenan, M.D.

Nasser K. Altorki, M.D.

F1. Increase in Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Expression in Esophageal Adenocarcinoma and Barrett's Esophagus.

Reginald V. Lord*, Ji Min Park*, Kathleen D. Danenberg*, Tom R. DeMeester, Jeffrey H. Peters*, Dennis Salonga*, Steven R. DeMeester*, Jeffrey A. Hagen*, Stefan Oberg*, Jon Singer*, Cedric G. Bremner* and Peter V. Danenberg*, Los Angeles, California

OBJECTIVE: To determine the role of the angiogenesis factors Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF) in the development and progression of Barrett's esophagus and adenocarcinomas of the esophageal body and gastroesophageal junction.

METHODS: VEGF and bFGF mRNA expression levels, relative to the stably expressed internal control genefi-actin, were measured in specimens of Barrett's intestinal metaplasia (IM, n=21), dysplasia (n=11), adenocarci-noma (n=17), and matching normal squamous esophagus tissues. A quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was used (ABI 7700 Sequence Detector (Taqman® system)).

RESULTS: Expression levels of both VEGF and bFGF were significantly increased in adenocarcinoma compared to either Barrett's esophagus or normal esophageal mucosa (table). Compared to normal mucosa, VEGF expression was significantly increased in non-dysplastic Barrett's mucosa, while bFGF expression was significantly increased in dysplastic Barrett's mucosa (all Mann-Whitney U test).

CONCLUSIONS: Angiogenesis factors are significantly upregulated in esophageal body and gastroesophageal junction adenocarcinomas and are likely to be important in the development of these cancers. Increased expression of angiogenesis factors can also be found in some patients with Barrett's esophagus, suggesting that Barrett's epithelium can have cellular migratory potential at an early stage. Expression levels of these genes may be useful markers for cancer detection in patients with Barrett's esophagus.

	VEGF	bFGF
CA vs. Normal	<0.0001	0.001
CA vs.IM	0.039	0.001
CA. vs. dysplasia	0.019	0.4

*By Invitation

F2. Sequential 5-Aza-2' Deoxycytidine (DAC)/Depsipeptide (DP) Treatment of Esophageal Cancer Cells Facilitates Their Recognition by Cytolytic T Cells (CTL) Specific for NY-ESO-1

Todd S. Weiser*, Galen Ohrunacht*, Julie Hong*, R-F Wang*, Dao M. Nguyen* and David S. Schrump, Bethesda, Maryland

OBJECTIVE: Previously we demonstrated synergistic enhancement of MAGE-3 expression and apoptosis in lung and esophageal cancer cells by sequential exposure to the demethylating agent 5-Aza-2' deoxycytidine, and the histone deacerylase inhibitor, Depsipeptide. This study was performed to determine if similar treatment could induce expression of NY-ESO-1 in esophageal cancer cells, and enable their recognition by CTL specific for this tumor antigen.

METHODS: Five esophageal cancer cell lines (all negative for NY-ESO-1 expression) were exposed to normal media, DAC, DP, or sequential DAC/ DP treatment using optimized induction regimens. Quantitative RT-PCR techniques were utilized to determine NY-ESO-1 mRNAcopy number relative to β-actin. Class I HLA expression was evaluated by PCR and flow cytometry techniques. TE-12 cells were transduced with a retroviral HLA-A31 construct or a retroviral control vector to examine recognition by HLA-A31 restricted NY-ESO-1 specific CTL utilizing y-IFN release assays.

RESULTS: Relative to baseline, NY-ESO-1 expression was enhanced 81-fold (range 11.8-198) by DAC alone (0.1 µM x 72h), 2.2-fold (0.8-4.1) by DP alone (25 ng/ml x 6h), and 139-fold (24.4-428) by sequential DAC/DP treatment. HLA expression was not altered under these conditions. CTL-mediated y-IFN release was observed in response to HLA-A31 transduced TE-12 cells following exposure to DAC (139 pg/ml) or DAC/DP (140 pg/ ml) but not normal media (0 pg/ml) or DP alone (0 pg/ml). Cytokine release in response to HLA-A31 transduced TE-12 cells following DAC or sequential DAC/DP treatment approximated that observed in response to autologous melanoma cells (157 pg/ml).

CONCLUSIONS: Sequential DAC/DP treatment represents a novel strategy to augment antitumor immunity in esophageal cancer patients.

*By Invitation

F3. Adenoviral Mediated Bak Gene Transfer Induces Apoptosis in Mesothelioma Cell Lines

Abujiang Pataer*, W. Roy Smythe*, Singling Fang*, Timothy J. McDonnell*, Jack A. Roth and Stephen G. Swisher*, Houston, Texas

OBJECTIVE: Mesothelioma cell lines are resistant to the induction of apoptosis following p53 gene therapy. We, therefore, evaluated the novel approach of adenoviral gene transfer of the pro-apoptotic Bcl-2 family member: Bak.

METHODS: Binary adenoviral Bak (Ad-GTBak +Ad-GV16) and Lac-Z (Ad-GTLac-Z+Ad-GV16) vectors were constructed for transduction of the mesothelioma cell lines: Ren and 145.

RESULTS: High levels of Bak gene transfer were seen following coadministration of Ad-GTBak and AdGV16 in both mesothelioma cell lines. High levels of apoptosis were induced 24 hours after Bak gene transfer with characteristic morphologic changes, Caspase 3 cleavage and subdiploid populations on FACS analysis (Table 1): Cell viability was decreased 48 - 72 hours after Bak gene transfer (Figure 1):

CONCLUSIONS: Adenoviral mediated overexpression of the Bak gene induces apoptosis and decreased cellular viability in mesothelioma cells. These data suggest that the gene transfer of pro-apoptotic Bcl-2 family members may represent a novel gene therapy strategy to treat mesothelioma.

Table 1: Percent Apoptosis of Mesothelioma Cell Lines
Mesothelioma Cell Line	PBS Control	Ad-GTLac-Z	Ad-GTBak
I45 (p53 wild-type)	1%	1%	36%*
REN (p53 mutant)	2%	3%	25%*

* p < 0.05

*By Invitation

F4. Differential Display Analysis Identifies New Highly Activated Gene in Mesothelioma: the Folate Receptor.

Raphael Bueno*, Krishnarao Appasani*, Harriet Mercer* and David J. Sugarbaker, Boston, Massachusetts

OBJECTIVE: The Folate Receptor is activated in certain solid tumors such as breast, renal and testicular carcinomas. As a consequence, antifolate drugs are effective in these neoplasms as they are in malignant pleural mesothelioma. We have, therefore, studied mesothelioma specimens to evaluate the Folate Receptor expression in this neoplasm.

METHODS: RNA was prepared from fresh tissues obtained from patients with mesothelioma. Differential display analysis was performed using normal lung RNA, normal pleura RNA, and tumor RNA. The analysis yielded 60 differentially expressed genes which were then characterized. One of the genes that was overexpressed in mesothelioma versus normal tissue was the Folate Receptor gene. In order to better characterize the expression of the Folate Receptor gene in mesothelioma, we performed in-situ hybridization utilizing antisense probes based on the sequence of the Folate Receptor. Frozen sections from 37 different patients(21 epithelial, 14 mixed, 2 sarcomatoid tumors)with pleural mesothelioma were analyzed using this technique. The controls included normal pleura, normal lung, and sense probes for all of the rumors. We also tested tumors known to have high expression of the Folate Receptor such as breast cancer, testicular cancer, and fallopian tube cancer as controls.

RESULTS: 31 of the 37 tumor specimens had between two- and four-fold higher Folate Receptor mRNA expression when compared with the control tissues. The slides of the other tested rumors demonstrated a similar elevation in the expression of the Folate Receptor. 6 tumors (4 epithelial and 2 mixed) were found to have the same or lower Folate Receptor mRNA expression.

CONCLUSIONS: The majority of mesothelioma tumors examined were found to express the Folate Receptor at two- to four-fold higher levels compared with normal pleura or lung. This is the first report of Folate Receptor overexpression in mesothelioma. This finding may explain the reports of up to a 30% clinical response to the antifolate drug methotrex-ate in mesothelioma. Furthermore, it encourages the testing of newer antifolate agents in the rational treatment of mesothelioma.

*By Invitation

F5. Obliterative Airway Disease Requires Direct Allorecognition

Wilson Y. Szeto*, Alyssa M. Krasinskas*, Daniel Kreisel*, Sicco H. Popma* and Bruce R. Rosengard*, Philadelphia, Pennsylvania

OBJECTIVE: Obliterative bronchiolitis (OB)is the primary cause of late death after lung transplantation. In a murine model, heterotopic tracheal allografts develop Obliterative airway disease (OAD), which resembles OB histologically. Chimeric tracheas bearing recipient-type antigen presenting cells (APCs) were used to examine whether direct allorecognition triggered by donor-type APCs is required for the development of OAD.

METHODS: Chimeric tracheas (i.e. tracheas having parenchyma and APCs of differing genotypes) were created via bone marrow transplantation (BMT). Tracheal segments from naive B6 mice, C3H→B6 chimeras, B6 Class I (B6^I-) or B6 Class II MHC antigen knockout mice (B6^II-) were transplanted subcutaneously in the dorsum of the recipients. The grafts were harvested at days 14 and 28, and the degree of luminal occlusion was determined by light microscopy.

RESULTS: At day 28, near complete occlusion was seen in all groups except for the chimeric tracheas, which lack donor-type APCs and, therefore, have substantial reduction of direct allorecognition. All isografts showed minimal luminal occlusion at both time points.

CONCLUSIONS: OAD is minimal in chimeric tracheas lacking donor-type APCs, but develops in the absence of either Class I or Class II MHC antigens. These findings suggest that direct allorecognition, triggered by donor APCs, is required for OAD, and that either the CD4⁺ or CD8⁺ direct allorecognition pathway is sufficient to initiate the process.

Tracheal	Tracheal	Tracheal		% Luminal Occlusion
Donor	Parenchyma	APCs	Reciplent	Day 14	Day 28
C3H	C3H	C3H	C3H	0% (n=2)	0% (n=3)
naive B6	B6	B6	C3H	not done	87.5% (n=4)
C3H→B6	B6	C3H	C3H	3% (n=3)	24% (n=5)
B6I-	B6I-	B6I-	C3H	0% (n=1)	100% (n=2)
B6II-	B6II-	B6II-	C3H	100% (n=2)	100% (n=3)

*By Invitation

F6. Adenovirus-Mediated Gene Transfer of Human Interleukin-10 Ameliorates Reperfusion Injury of Rat Lung Isografts

Hideki Itano*, Wanjiang Zhang*, Thalachallour Mohanakumar* and G. Alexander Patterson, St. Louis, Missouri

OBJECTIVE: Interleukin-10 has been identified as a potent inhibitor of various inflammatory responses. The objective of this study was to examine the feasibility of human interleukin-10 (hIL-10) gene transfer into rat lung isografts and its effect on subsequent ischemia reperfusion (I/R) injury.

METHODS: Male F344 rats were divided into four groups and underwent left lung isotransplantation. Twenty four hours prior to harvest, 5 x 10E9 pfu (Group I, n=6) or 1 x 10E10 pfu (Group II, n=7) of AdRSVhIL-10 was intravenously administered to donor rats. In Group I-C (n=6) and Group II-C (n=6), serving as control, 5 x 10E9 pfu and 1 x 10E10 pfu of AdCMVLacZ were administered respectively. In all groups, grafts were preserved forlS hours at 4 degree prior to implantation, and assessed 24 hours after reperfusion. Transgene expression of hIL-10 was assessed by both RT-PCR and immunohistochemistry (IHC). Graft iNOS mRNA expression was assessed by RT-PCR. Isograft gas exchange and MPO activity were also assessed.

RESULTS: Dose-dependent transgene expression was detected by RT-PCR (p < 0.05) and IHC. Gas exchange in Groups I and II was significantly better than in Groups I-C and II-C (Table). MPO activity (OD/min/ng protein) in Group II was significantly lower than in Group II-C (0.082 ±0.034 vs. 0.117 ±0.028, p < 0.05). The iNOS mRNA expression in Group II was significantly lower than in Group II-C (0.332 ± 0.39 vs. 0.745 ± 0.29, p< 0.05).

CONCLUSION: In vivo lung isograft adenovirus-mediated hIL-10 gene transfer ameliorates I/R injury.

Isograft gas exchange

Groups	PaO2	PaCO2	Groups	PaO2	PaCO2
Group 1	164.72 ±85.3*	33.40 ± 6.80*	Group II	153.19±113*	43.64 ±15.1
Group I-C	82.37±19.1	51.23 ± 11.9	Group II-C	77.95 ±33.4	50.60 ±17.7

* p < 0.05 vs. control, (mmHg)

*By Invitation

F7. SCRlslex Is Superior to Scrl in Reducing Ischemia/Reperfusion Injury in Experimental Lung Transplantation

Uz Stammberger*, Sven Hillinger*, Giovanni L. Carboni*, Walter Weder*, Juerg Hamacher* and Ralph A. Schmid*, Berne and Zurich, Switzerland

OBJECTIVE: The nonspecific immune response including activation of the complement system and polymporphonuclear leukocytes (PMN) is critical for the mediation of reperfusion injury. We investigated the combined blockade of the complement system and leukocyte adhesion by a novel drug, sCR1sLex. In this glycoprotein, the sugar molecules of soluble complement receptor type 1 (sCR1; CD35) are substituted with the selectin ligand sialyl LewisX (sLex; CD15s)(AVANTIMMUN, Needham MA).

METHODS: Orthotopic allogeneic single left lung transplantation was performed in rats (BN to F344) after 20 hours ischemia. Three groups (n=5) were studied: Group I (control), Group II received sCRl (10 mg/kg) and group III sCRlsLex (10 mg/kg) 15 min prior to reperfusion by intracar-diac injection. Twenty-four hours after reperfusion, the native contralat-eral lung was occluded to assess isolated gas exchange of the graft. In additional animals (n=5/group), lung tissue was frozen 24 h after reperfusion and assessed for myeloperoxidase activity (MPO; neutrophil migration), and thiobarbituric acid reactive substances (TEARS; lipid peroxidation).

RESULTS: Graft function in group III was superior to group I and group II:

CONCLUSIONS: Our data indicate that combined inhibition of the complement system and leukocyte adhesion with sCR1sLex reduces reperfusion injury significantly, and that both mechanisms are effectively inhibited in this model.

Results

Group	PaO2 [mmHg]	_MPO [ΔOD/mg/min]	TBARS [pmol/g]
I	56±7	1.0+±0.09	10.6±0.54
II	243±45**	0.48±0.07***	8.32+0.89*
III	383±53***#	0.33±0.05***	6.210.37***#
Native lung		0.2210.05#	3.9±0.75*#

*p<0.05,**p^#p<0.05vs. sCR1

*By Invitation

F8. Manganese Superoxide Dismutase Gene Insertion Protects Normal Cells During Photodynamic Therapy

Hsien Yean Wong*, Micheal Epperly*, Tony Godfrey*, Joel Greenberger* and James D. Luketich*, Pittsburgh, Pennsylvania

OBJECTIVE: PDT is a new modality for the treatment of esophageal and lung cancers which involves free radical mediated cell death leading to mitochondrial disruption and apoptosis. PDT efficacy is limited by damage to surrounding normal cells. MnSOD localizes to mitochondria and scavenges free radicals. Our Objective was to determine if MnSOD overexpression would protect against PDT-induced cell death.

METHODS: Normal mouse hematopoetic cell line (32 D c!3) served as control. Two subclones transfected with human MnSOD transgene (2C6, 1F2) were produced which overexpress MnSOD. Photofrin (lug/ml) was added to cell culture 24 hrs before light activation. Survival was determined by plating cells in methylcellulose and colony counting. Staining for apoptosis was performed.

RESULTS: PDT dose response curve slope (D₀) for 32D c!3, 2C6 and 1F2 were 2.8,13, and 6.7 indicating better survival for MnSOD incorporation. Percent cell kill was determined by apoptotic staining (see table).

CONCLUSIONS: These results suggest MnSOD gene incorporation protects normal cells from PDT-induced death. Further work by our groups in ongoing to determine if MnSOD gene incorporation leads to overexpression and protection in the normal human esophagus.

Mean Apoptosis (%)

Light exposure (J)	32 D	2C6	1F2
4	2.5	8	0.0
6	14	6.5	15
7	19	2.0*	22
8	58	4.0*	38*

* indicates significant resistance to apoptosis compared to 32D, p < 0.05

*By Invitation

F9. Lung Volume Reduction Surgery Restores the Normal Diaphragmatic Length-Tension Relationship in Emphysematous Rats

Joseph B. Shrager*, Dong-Kwan Kim*, Yahya Hashmi*, Hansell H. Stedman*, Sanford Levine* and Larry R. Kaiser, Philadelphia, Pennsylvania

OBJECTIVE: Improved respiratory muscle function is a major effect of lung volume reduction surgery (LVRS). There has been no previous study of respiratory muscle in an experimental model of LVRS. We sought to elucidate the mechanism by which diaphragmatic function improves following LVRS.

METHODS: We developed a model of elastase-induced emphysema and LVRS via median sternotomy (MS) in rats. Five months following emphysema induction, lung volume (VC) was determined in intubated, anesthetized emphysema and control animals. Costal diaphragmatic length (LC) was measured in vivo, and Lo (the length at which maximal twitch force is generated) was determined in vitro. Also 5 months following elastase administration, a cohort underwent LVRS or sham MS. Five months following operation, these animals were similarly studied.

RESULTS: VC was increased in emphysematous rats versus controls (50.9+/-4.8 vs 45.4+/-3.8cc, p=.024). VC was decreased in emphysematous animals which had undergone LVRS versus sham MS (44.7+/-2.1 vs 49.4+/-2.7cc, p=.001). LC (1.99+/-.11 vs 2.24+/-.11cm, p=.001) and Lo (2.25+/-.21 vs 2.48+/-.29cm, p=.038) were shorter in emphysematous than controls animals. Following LVRS, LC (2.13+/-.07 vs 1.83+/-.16cm, p<.001) and Lo (2.50+/-.17 vs 2.27+/-.15cm, p=.013) were longer than in sham MS animals.

CONCLUSIONS: In this experimental model of emphysema and LVRS, emphysema shortens Lo and shifts the diaphragmatic length-tension curve to the left; LVRS returns Lo toward normal and shifts the diaphragmatic length-tension curve back to the right. This restoration toward normal physiology may enable the improvement in diaphragmatic function seen following LVRS. The mechanism by which Lo lengthens merits further investigation.

§Author has a relationship with Metabolix, Inc.

*By Invitation

F10. Replacement of the Trachea with an Autologous Aortic Graft

Emmanuel Martinod*, Rachid Zegdi*, Gilbert Zakine*, Paul Fornes*, Alexandre D'Audiffret*, Juan-Carlos Chachques*, Jacques Azorin* and Alain Carpentier, Paris and Bobigny, France.

OBJECTIVE(s): Tracheal reconstruction after extensive resection remains an unsolved surgical problem. Many studies have evaluated various substitutes including prostheses, tracheal allografts or autologous grafts with disappointing results. The goal of this experimental study was to evaluate the replacement of a large segment of the trachea using an autologous aortic graft, selected for specific advantages: similar diameter, resistance to infection and lack of immune response.

METHODS: In 20 sheep, a 5 cm segment of the cervical trachea was resected and replaced by a 5 cm segment of the descending thoracic aorta. The thoracic aorta was reconstructed with a vascular prosthesis using an arterial shunt. A definitive Ultraflex TM stent (n=13) or a temporary Novatech TM stent (n=7) was placed in the lumen of the aortic graft to prevent collapse. Clinical, bronchoscopic and histologic examinations were performed at 1, 3, 6, 9 and 12 months.

RESULTS: All animals survived the operation and there were no paraplegia. There has been only 3 complications : one stent displacement, one laryngeal edema, one infection. In the remaining 17 animals, there was no anastomotic leakage, rupture or stenosis. Histology showed a progressive transformation of the arterial segment into a tracheal tissue including a neoformation of cartilage and a continuous epithelium.

CONCLUSIONS: This study shows that an autologous aortic graft could be a valuable substitute for tracheal replacement. In humans, the abdominal aorta could be used and replaced with a prosthesis, a minor inconvenience compared to the benefit of a durable tracheal reconstruction.

*By Invitation

8:00 a.m. ADULT CARDIAC SURGERY FORUM SESSION

Constitution Hall, Metro Toronto Convention Centre

Moderators: Andrew S. Wechsler, M.D.

Fred A. Crawford, M.D.

F11. Marrow Stromal Cells for Cellular Cardiomyoplasty: Feasibility and Clinical Advantages

Jih-Shiuan Wang*, Dominique Shum-Tim*, Jacques Galipeau*, Edgar Chedrawy*, Nicoletta Eliopoulos* and Ray Chu-Jeng Chiu, Taipei, Taiwan ROC; Montreal, PQ, Canada.

OBJECTIVE: Bone marrow stromal cells (MSC) are mesenchymal stem cells able to differentiate into cardiomyocytes in vitro. We tested the hypothesis that MSC, when implanted into myocardium, can undergo milieu-dependent differentiation and form long-term, incorporated grafts expressing cardiomyogenic phenotypes in vivo.

METHODS: Isogenic adult rats were used as donors and recipients to simulate autologous transplant in patients. MSC isolated from donor leg bones were expanded in culture, labeled with DAPI (4', 6-diamidino-2-phenylindole), and then injected directly into the myocardium of the recipients. The hearts were harvested from 4 days to 12 weeks after implantation, and the implant sites were sectioned for histological and immuno-histochemical studies to identify the phenotypes of the labeled cells.

RESULTS: Viable DAPI labeled cells can be identified in host myocardium at all time points after implantation. The implanted cells can be seen to juxtapose to the host myocardium, or pooled together at the implantation area. At the periphery of implantation area and within the adjacent host myocardium, DAPI labeled cells appear to be incorporated into the host myofibers and join with myocytes not labeled by DAPI. Such DAPI labeled cells showed positive stain for sarcomeric myosin heavy chain (Using MF20 antibody) and connexin 43 (Gap junction protein in the intercalated disks) by 4 weeks after implantation.

CONCLUSIONS: Fetal and "altered" adult cardiomyocytes, as well as skeletal myoblasts had been used as donor cells for cellular cardiomyoplasty, and shown to improve the function of impaired ventricles. Our findings indicate that MSC can also be used as donor cells, and in appropriate microenvironment, they can exhibit cardiomyogenic phenotypes, and may replace native cardiomyocytes lost by necrosis or apoptosis. However, in contrast to other cell sources, autologous MSC can be obtained repeatedly by simple routine bone marrow aspiration, and expanded vastly in vitro prior to implantation. Furthermore since autologous implants will not require immunosuppression, clinical use of MSC for cellular cardiomyoplasty appears to be most advantageous.

*By Invitation

F12. Cellular Therapy Reverses Myocardial Dysfunction

Juan C. Chachques*, Charissa Rajnoch*, Alain Berrebi*, Nicolas Borenstein*, Ming Shen*, Nicola D'Attellis*, Jean N. Fabiani*, Alain F. Carpentier, Paris, France.

OBJECTIVE(s): The aim of cell transplantation into pathologic myocardium is to repair, replace or enhance the biological function of the altered ventricle, restoring a functional myocardial mass, and hence improving the contractile performance of the heart.

METHODS: Autologous myoblasts obtained from skeletal muscles were implanted into the LV wall in an experimental model of partial ventricular akinesia. Step I: Chronic cardiac deficiency was developed by local injection (3 mg/1.5 ml) of snake cardiotoxin (C 9759, Sigma Chem.) in the LV wall of sheep, through a left mini-thoracotomy. Step II: Autologous myoblasts taken from skeletal muscle biopsy of animal limbs were cultured for 3 weeks. A selection procedure was performed to obtain a pure culture. Then, through a sternotomy, myoblasts were introduced in the injured myocardium. A cell suspension of 2 x 10⁷ cells diluted in 500mL of Ham-F12 medium was injected. Echocardiographic studies (Color Kinesis H.P.) were performed after toxin, after cell injections and at 2 months. Histopathologic studies were carried out at 2 months. Control groups consisted of injection of toxin alone (6 sheep) and toxin + culture medium (6 sheep).

RESULTS: All animals survived. After toxin injections, serum levels of troponin increased significantly (up to 125±16 ng/ml), and LV wall morion (regional fraction area change: RFAC) decreased from 71±4 to 40±2.5%, p<0.05. Two months following myoblasts implantation, ventricular remodeling partially reversed and myocardial contractility significantly recovered in the cell implanted group (RFAC: 65±7%). Healthy myoblasts were observed in 75% of myocardial histological studies. Cardiotoxin administration generated transmural necrosis, with severe damage of cardiomyocytes and interstitial tissues (myocardial matrix).

CONCLUSIONS: Myoblast implantation was associated with the recovery of myocardial contractility in an experimental model of segmentary ventricular akinesia ("infarct-like" myocardial lesion). Healthy myoblasts were observed 2 months after myocardial implantation. Further studies on host-cell interactions (mechanical and electrical coupling) are necessary.

*By Invitation

F13. Hemodynamic Unloading Leads to Regression of Pulmonary Vascular Disease in Rats

Stacy B. O'Blenes*, Stefan Fischer*, Brendan Mclntyre*, Shaf Keshavjee, Marlene Rabinovitch*, Toronto, ON, Canada.

OBJECTIVE: Treatment options for patients with pulmonary vascular disease secondary to a congenital heart defect are still mainly limited to heart-lung transplantation or lung transplantation with repair of the cardiac lesion. However, we have previously shown that the structural changes associated with pulmonary hypertension can be reversed by stress unloading in an organ culture model. We now test the hypothesis that hemodynamic unloading will lead to regression of pulmonary vascular disease in the intact animal.

METHODS: Right middle+lower lobectomy and monocrotaline injection was performed in Lewis rats (n=22) to cause pulmonary vascular disease from a combined hemodynamic and toxic injury. Twenty eight days later the left lungs were examined (n=10) or exposed to normal pulmonary artery (PA) pressure for an additional 14 (n=5) or 28 (n=7) days by transplantation into healthy recipients. PA pressure, ventricular weight, and PA morphology was evaluated in each group.

RESULTS: Pulmonary hypertension (50 vs. 16 mmHg, p<0.001) and right ventricular hypertrophy (RV/LV weight 0.69 vs. 0.32, p<0.001) associated with PA medial hypertrophy (28.2 vs. 7.2 % of wall thickness, p<0.001) and muscularization of small PAs (92.3% vs. 19.4%, p<0.001) developed by day 28 (compared to untreated controls). However, transplantation into healthy recipients effectively unloaded the lungs (mean PA pressure 17 and 25 mmHg at 14 and 28 days post transplant) and resulted in progressive normalization of medial hypertrophy (15.6 and 12.1% at 14 and 28 days) and muscularization (65.1 and 42.2% at 14 and 28 days) relative to untransplanted controls (p<0.005 in each case).

CONCLUSIONS: Hemodynamic unloading of lungs with pulmonary vascular disease results in progressive normalization of PA structure. These results are the first to provide a rationale for attempting to induce regression of pulmonary vascular disease by pressure unloading of the pulmonary circulation. PA banding should be critically evaluated as a strategy for staged surgical repair of congenital heart defects despite presumed irreversible pulmonary hypertension.

*By Invitation

F14 Cerebral Effects of Reperfusion After Hypothermic Circulatory Arrest

Marek P. Ehrlich*, Donald Weisz*, David Wolfe*, Carol A. Bodian*, Ning Zhang*, Jock N. McCullough* and Randall B. Griepp, New York, New York

OBJECTIVE: This study was undertaken to explore whether an interval of cold reperfusion can improve cerebral outcome following prolonged hypothermic circulatory arrest (HCA).

METHODS: Sixteen pigs (27-30 kg) underwent 90 minutes of HCA at a brain temperature of 20C. Eight animals were rewarmed immediately after HCA; eight were reperfused for 20 minutes at 20C and then rewarmed. Electrophysiological recordings, fluorescent microsphere determinations of cerebral blood flow (CBF), calculations of cerebral oxygen consumption (CMRO2), and direct measurements of intracranial pressure (ICP, mm Hg) were obtained at baseline (37C), before HCA, after cardiopulmonary bypass (CPB) was restarted, and at 2, 4, and 6 hours thereafter. Histopathology and wet/dry brain weight were determined after sacrifice.

RESULTS: CBF and CMRO2 decreased during cooling: CMRO2 returned to baseline levels after 4 hours, but CBF remained depressed until 6 hours in both groups. Cold reperfusion failed to improve electrophysiological recovery or to reduce brain weight, but the increase in median ICP usually seen after prolonged HCA was significantly less after cold reperfusion than after immediate rewarming (p = 0.02),), as seen in the table below. Although no significant difference in the incidence of cerebral histopathology between groups was found, all three animals with ICP > 15 following immediate rewarming had histopathological lesions, and high ICP was more prevalent among all animals with subsequent histopathology (p = 0.03).

CONCLUSIONS: Cold reperfusion significantly inhibited the rise in ICP usually seen after 90 minutes of HCA at 20C, suggesting that it may decrease cerebral edema and thereby improve outcome following prolonged HCA.

Intracranial Pressure (ICP, mmHg)
	Before HCA	After CPB	2 Hours	4 Hours	6 Hours
Immediate Rewarming	9.5	13	15	15	14.5
Cold Reperfusion	9	9	10	11.5	12.5

*By Invitation

F15. The Fate of a Tissue Engineered Cardiac Graft in the Right Ventricular Outflow Tract

Tetsuro Sakai*, Ren-Ke Li*, Richard D. Weisel, Donald A. G. Mickle*, Eung Joong Kim*, Zhi-Qian Jia*, Terrence M. Yau*, Toronto, ON, Canada.

OBJECTIVE: Currently available graft materials for repair of cardiac defects are nonviable and contribute to late morbidity and mortality. We have developed a beating cardiomyocyte-seeded biodegradable graft in vitro. We evaluated the in vivo fate of this graft in the right ventricular outflow tract (RVOT) of adult rats.

METHODS: Cultured fetal or adult rat cardiomyocytes (1 x 10⁶ cells) were seeded into a gelatin mesh (15 x 15 x 1 mm) and maintained in tissue culture for 1 or 3 weeks. 15% of the cells were prelabelled with BrdU prior to graft seeding, to permit cell identification after graft implantation. The RVOT free wall of syngeneic adult rats was partially resected and replaced with grafts seeded with fetal or adult cardiomyocytes, or unseeded grafts (N=10 per group). The hearts were excised at 4 or 12 weeks, and the grafts and perigraft tissue examined histologically. Graft endothelialization was evaluated by immunostaining for Factor VIII, and persistence of seeded cells by staining for BrdU.

RESULTS: Mean endocardial surface area of the grafts 4 weeks after implantation was 8.8±3.0 mm², which represented 6.7±2.9% of the area of the RV free wall. Factor VHI staining confirmed complete endothelialization of the graft surface at 4 weeks. A significant perigraft lymphocytic infiltrate was noted. By 12 weeks after implantation, the original gelatin framework of the graft had been completely resorbed. BrdU positive cells were noted throughout the graft, but cell numbers decreased with time. Unseeded grafts were completely replaced by fibrous tissue. Graft thickness decreased significantly between 4 and 12 weeks in the unseeded (p=0.003), fetal (p=0.0001) and adult (p=0.07) cardiomyocyte groups, but to a similar degree (p=0.2).

CONCLUSIONS: The RVOT of adult rats can be replaced with a cardiomyocyte-seeded biodegradable graft. The seeded cells persist within the graft over 12 weeks, but cell survival is limited by the host inflammatory response to the gelatin substrate. A less antigenic substrate may enhance cell survival. These techniques may lead to the development of a viable, cellular graft with growth potential for reconstruction of congenital cardiac defects.

*By Invitation