- Email: [email protected]
94: The Inhibitor of Apoptosis Birc6 Is a Sensitive and Specific Marker of Cardiac Allograft Rejection
The Journal of Heart and Lung Transplantation Volume 27, Number 2S
94 The Inhibitor of Apoptosis Birc6 Is a Sensitive and Specific Marker of Cardiac Allograft Rejection S. Aharinejad,1,2 O. Andrukhova,2 R. Schaefer,2 K. Krenn,2,3 C. May,2 A. Zuckermann,1 D. Zimpfer,1 M. Grimm,1 1Dept. of Cardiothoracic Surgery, Medical University of Vienna, Vienna, Austria; 2Lab. for Cardiovasc. Research, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria; 3 Dept. of Internal Medicine II, Medical University of Vienna, Vienna, Austria Purpose: Birc6 inhibits apoptosis by facilitating the degradation of pro-apoptotic molecules. RNF41 is an ubiquitin ligase that promotes the ubiquitination and degradation of Birc6 in response to apoptotic stimuli. Birc6 degradation and induction of apoptosis is induced in RNF41-expressing but not RNF41-deficient cells. Whether the Birc6/ RNF41 system is functional in cardiac tissue and plays a role in allograft rejection-induced cell death is unknown. Methods and Materials: Birc6 and RNF41 gene expression was examined by real time RT-PCR measurements in endomyocardial biopsies of transplanted hearts at 1, 2, 3, 4, 7, 12, 24 and 52 weeks post-transplantation. Graft rejection was diagnosed and scored in 170 patients and 1360 biopsies by histology according to ISHLT guidelines. Results: Birc6 myocardial gene expression was not significantly different at rejection grade 1R compared to grade 0 (p⬍0.65). Birc6 myocardial gene expression was almost completely abolished at rejection grades 2R and 3R (p⬍0.001). In contrast, RNF41 gene myocardial expression was consistently upregulated at rejection grades 1R-3R compared to rejection grade 0 (p⬍0.01). Conclusions: These results suggest for the first time that the Birc6/RNF41 system is operating to mediate acute cell death in cardiac allograft rejection. These results also indicate that high mRNA levels of RNF41 initiate acute cell death by eliminating Birc6 gene expression levels in high-grade allograft rejection (rejection grades 2R and 3R) but not at grades 0 or 1R. Real time RT-PCR measurements of Birc6 can be performed quickly, are specific and relatively inexpensive. Assessment of Birc6 levels by this method could therefore be an accurate and quantitative method to diagnose cardiac allograft rejection. 95 Clinical Experience with Recombinant Factor VII Use in Patients with Left Ventricular Assist Devices B.A. Bruckner,1 L.P. Jacob,2 J.C. Walkes,1 O. Habib,1 M.J. Reardon,1 M. Loebe,1 I.D. Gregoric,2 O.H. Frazier,2 1DeBakey Heart Center, The Methodist Hospital, Houston, TX; 2Transplant & Assist Devices, Texas Heart Institute, Houston, TX Purpose: Recombinant Factor VII (rFVIIa) is a powerful hemostatic agent that can be used in patients with refractory bleeding undergoing cardiothoracic procedures. However, its use in patients with left ventricular assist devices (LVADs) represents a challenge with the potential for increased risk of thromboembolic events. Methods and Materials: A total of 60 patients underwent LVAD related procedures and received rVIIa for perioperative bleeding. Laboratory values, transfusion requirements and thromboembolic events were recorded before and after rFVIIa administration. Results: 60 patients (50 men, 10 women; mean age, 55 yrs) underwent a total of 60 LVAD related procedures including 51 LVAD insertions and 9 pump exchange procedures. Due to refractory bleeding, rFVIIa was administered to all patients immediately following surgery either in the operating room (n⫽40) or the ICU (n⫽20). The dose ranged from 10-20 mcg/kg in 30 patients and 30-70 mcg/kg in the remaining 30 patients. Administration of
Abstracts
S93
rFVIIa resulted in significant decreases in PT (15.7 sec ⫾ 6.4 vs 12.5 sec ⫾3.1, p⬍0.001) and aPTT (58.2 sec ⫾ 32.8 vs 51.4 sec ⫾32.6, p⫽0.003). Hemoglobin (gm/dL) levels increased (9.4⫾2.2 vs 10.2⫾1.6, p⬍0.001). Transfusion requirements significantly decreased including packed red blood cells (21.5u ⫾13 vs 10.2u ⫾8.9, p⬍0.001) and fresh frozen plasma (25.1u ⫾20.6 vs 7.7u ⫾6.2, p⬍0.001). There were 15 (25%) thromboembolic events recorded including DVT(n⫽2), pulmonary embolus (n⫽3), cerebral vascular accident (n⫽3), myocardial infarction (n⫽2), cardiac thrombus (n⫽2), and peripheral arterial occlusion (n⫽3). The lower dose group (10-20 mcg/kg) had fewer thrombotic events when compared to the higher dose group (6.7% vs 43%). Conclusions: Administration of rVIIa can be effective in controlling life-threatening bleeding in patients undergoing LVAD procedures. Due to the higher risk of thrombotic events in these patients, lower doses should be used until prospective studies can detect the relationship between rFVIIa dosage and incidence of thromboembolic events.
96 Sustained Activation of Vascular Endothelium Induces the Tissue Factor Pathway in LVAD Recipients S.R. Panch,1 J. Hrabe,1 A. Solovey,1 C. Toninato,1 R.P. Hebbel,1 R. John,1 1University of Minnesota, Minneapolis, MN Purpose: Progress of LVAD therapy is limited by thromboembolic complications. To better understand coagulation pathophysiology, we studied endothelial and coagulation system changes in LVAD patients. Methods and Materials: Endothelial activation markers (circulating endothelial cells [CEC] with expressed E-selectin, ICAM and Tissue factor[TF]), thrombin generation (prothrombin fragments1,2[PF1⫹2] and thrombin antithrombin[TAT]), and fibrinolysis(D-dimer) were studied in 22 LVAD patients on day 0, post-op days 7, 90,180 and in 6 controls undergoing non-LVAD cardiac surgery. Results: Baseline values of ICAM, E-selectin, TF, TAT and D-dimer were significantly higher in LVAD patients.Thrombin generation and fibrinolysis markers peaked postoperatively and declined to ⱕ baseline levels by 90 days; endothelial activation markers increased likewise but continued to remain elevated up to 6 months. In controls, baseline measures were lower and declined significantly by day 7 as compared to LVAD patients (pvalue ⬍0.05). Conclusions: These findings suggest that persistent activation of vascular endothelium may be an important trigger for the tissue factor pathway activation, creating a sustained prothrombotic environment in LVAD patients. Further work elucidating the relationship between this prothrombotic mileu and thromboembolic complications is needed to optimize anticoagulation strategies. Test means(ⴞSEM) for LVAD patients Test
Normal
Day 0 (pre-op)
Day 7 (post-op)
Day 90
Day 180
CECs (cells/ml) E-selectin(%) ICAM(%) TF (%) TAT (uG/L) PF 1⫹2(nM/l) D-Dimer (FEU/ml)
2.6⫾0.6 (0-5) 2⫾5 25⫾15 10⫾13 1-4.1 0-0.22 ⬍0.5
3.7⫾0.9 54.2⫾4.4 55.1⫾4.3 57.8⫾4.5 10.2⫾2 0.3⫾0.03 2.1⫾0.4
6.1⫾1.4 73.2⫾2.9* 71⫾4.3* 78.5⫾3.5* 11.4⫾1.1 0.5⫾0.07* 5.9⫾0.7*
3.7⫾0.9 53.1⫾6.5 64.9⫾4.7 53⫾5 6.5⫾1.1 0.25⫾0.05 2.3⫾0.5
2.8⫾0.9 65.6⫾5.2 62.8⫾6.6 76⫾7.8 8.1⫾2.3 0.24⫾0.05 1.9⫾0.4
*p⬍0.05, paired t tests comparing post op values with baseline
97 Preoperative Parameters at the Time of Left Ventricular Assist Device Placement Predict the Need for Biventricular Mechanical Support
94 The Inhibitor of Apoptosis Birc6 Is a Sensitive and Specific Marker of Cardiac Allograft Rejection S. Aharinejad,1,2 O. Andrukhova,2 R. Schaefer,2 K. Krenn,2,3 C. May,2 A. Zuckermann,1 D. Zimpfer,1 M. Grimm,1 1Dept. of Cardiothoracic Surgery, Medical University of Vienna, Vienna, Austria; 2Lab. for Cardiovasc. Research, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria; 3 Dept. of Internal Medicine II, Medical University of Vienna, Vienna, Austria Purpose: Birc6 inhibits apoptosis by facilitating the degradation of pro-apoptotic molecules. RNF41 is an ubiquitin ligase that promotes the ubiquitination and degradation of Birc6 in response to apoptotic stimuli. Birc6 degradation and induction of apoptosis is induced in RNF41-expressing but not RNF41-deficient cells. Whether the Birc6/ RNF41 system is functional in cardiac tissue and plays a role in allograft rejection-induced cell death is unknown. Methods and Materials: Birc6 and RNF41 gene expression was examined by real time RT-PCR measurements in endomyocardial biopsies of transplanted hearts at 1, 2, 3, 4, 7, 12, 24 and 52 weeks post-transplantation. Graft rejection was diagnosed and scored in 170 patients and 1360 biopsies by histology according to ISHLT guidelines. Results: Birc6 myocardial gene expression was not significantly different at rejection grade 1R compared to grade 0 (p⬍0.65). Birc6 myocardial gene expression was almost completely abolished at rejection grades 2R and 3R (p⬍0.001). In contrast, RNF41 gene myocardial expression was consistently upregulated at rejection grades 1R-3R compared to rejection grade 0 (p⬍0.01). Conclusions: These results suggest for the first time that the Birc6/RNF41 system is operating to mediate acute cell death in cardiac allograft rejection. These results also indicate that high mRNA levels of RNF41 initiate acute cell death by eliminating Birc6 gene expression levels in high-grade allograft rejection (rejection grades 2R and 3R) but not at grades 0 or 1R. Real time RT-PCR measurements of Birc6 can be performed quickly, are specific and relatively inexpensive. Assessment of Birc6 levels by this method could therefore be an accurate and quantitative method to diagnose cardiac allograft rejection. 95 Clinical Experience with Recombinant Factor VII Use in Patients with Left Ventricular Assist Devices B.A. Bruckner,1 L.P. Jacob,2 J.C. Walkes,1 O. Habib,1 M.J. Reardon,1 M. Loebe,1 I.D. Gregoric,2 O.H. Frazier,2 1DeBakey Heart Center, The Methodist Hospital, Houston, TX; 2Transplant & Assist Devices, Texas Heart Institute, Houston, TX Purpose: Recombinant Factor VII (rFVIIa) is a powerful hemostatic agent that can be used in patients with refractory bleeding undergoing cardiothoracic procedures. However, its use in patients with left ventricular assist devices (LVADs) represents a challenge with the potential for increased risk of thromboembolic events. Methods and Materials: A total of 60 patients underwent LVAD related procedures and received rVIIa for perioperative bleeding. Laboratory values, transfusion requirements and thromboembolic events were recorded before and after rFVIIa administration. Results: 60 patients (50 men, 10 women; mean age, 55 yrs) underwent a total of 60 LVAD related procedures including 51 LVAD insertions and 9 pump exchange procedures. Due to refractory bleeding, rFVIIa was administered to all patients immediately following surgery either in the operating room (n⫽40) or the ICU (n⫽20). The dose ranged from 10-20 mcg/kg in 30 patients and 30-70 mcg/kg in the remaining 30 patients. Administration of
Abstracts
S93
rFVIIa resulted in significant decreases in PT (15.7 sec ⫾ 6.4 vs 12.5 sec ⫾3.1, p⬍0.001) and aPTT (58.2 sec ⫾ 32.8 vs 51.4 sec ⫾32.6, p⫽0.003). Hemoglobin (gm/dL) levels increased (9.4⫾2.2 vs 10.2⫾1.6, p⬍0.001). Transfusion requirements significantly decreased including packed red blood cells (21.5u ⫾13 vs 10.2u ⫾8.9, p⬍0.001) and fresh frozen plasma (25.1u ⫾20.6 vs 7.7u ⫾6.2, p⬍0.001). There were 15 (25%) thromboembolic events recorded including DVT(n⫽2), pulmonary embolus (n⫽3), cerebral vascular accident (n⫽3), myocardial infarction (n⫽2), cardiac thrombus (n⫽2), and peripheral arterial occlusion (n⫽3). The lower dose group (10-20 mcg/kg) had fewer thrombotic events when compared to the higher dose group (6.7% vs 43%). Conclusions: Administration of rVIIa can be effective in controlling life-threatening bleeding in patients undergoing LVAD procedures. Due to the higher risk of thrombotic events in these patients, lower doses should be used until prospective studies can detect the relationship between rFVIIa dosage and incidence of thromboembolic events.
96 Sustained Activation of Vascular Endothelium Induces the Tissue Factor Pathway in LVAD Recipients S.R. Panch,1 J. Hrabe,1 A. Solovey,1 C. Toninato,1 R.P. Hebbel,1 R. John,1 1University of Minnesota, Minneapolis, MN Purpose: Progress of LVAD therapy is limited by thromboembolic complications. To better understand coagulation pathophysiology, we studied endothelial and coagulation system changes in LVAD patients. Methods and Materials: Endothelial activation markers (circulating endothelial cells [CEC] with expressed E-selectin, ICAM and Tissue factor[TF]), thrombin generation (prothrombin fragments1,2[PF1⫹2] and thrombin antithrombin[TAT]), and fibrinolysis(D-dimer) were studied in 22 LVAD patients on day 0, post-op days 7, 90,180 and in 6 controls undergoing non-LVAD cardiac surgery. Results: Baseline values of ICAM, E-selectin, TF, TAT and D-dimer were significantly higher in LVAD patients.Thrombin generation and fibrinolysis markers peaked postoperatively and declined to ⱕ baseline levels by 90 days; endothelial activation markers increased likewise but continued to remain elevated up to 6 months. In controls, baseline measures were lower and declined significantly by day 7 as compared to LVAD patients (pvalue ⬍0.05). Conclusions: These findings suggest that persistent activation of vascular endothelium may be an important trigger for the tissue factor pathway activation, creating a sustained prothrombotic environment in LVAD patients. Further work elucidating the relationship between this prothrombotic mileu and thromboembolic complications is needed to optimize anticoagulation strategies. Test means(ⴞSEM) for LVAD patients Test
Normal
Day 0 (pre-op)
Day 7 (post-op)
Day 90
Day 180
CECs (cells/ml) E-selectin(%) ICAM(%) TF (%) TAT (uG/L) PF 1⫹2(nM/l) D-Dimer (FEU/ml)
2.6⫾0.6 (0-5) 2⫾5 25⫾15 10⫾13 1-4.1 0-0.22 ⬍0.5
3.7⫾0.9 54.2⫾4.4 55.1⫾4.3 57.8⫾4.5 10.2⫾2 0.3⫾0.03 2.1⫾0.4
6.1⫾1.4 73.2⫾2.9* 71⫾4.3* 78.5⫾3.5* 11.4⫾1.1 0.5⫾0.07* 5.9⫾0.7*
3.7⫾0.9 53.1⫾6.5 64.9⫾4.7 53⫾5 6.5⫾1.1 0.25⫾0.05 2.3⫾0.5
2.8⫾0.9 65.6⫾5.2 62.8⫾6.6 76⫾7.8 8.1⫾2.3 0.24⫾0.05 1.9⫾0.4
*p⬍0.05, paired t tests comparing post op values with baseline
97 Preoperative Parameters at the Time of Left Ventricular Assist Device Placement Predict the Need for Biventricular Mechanical Support