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Noninvasive. Cost-effective detection of cardiac allograft vasculopathy by electron beam computed tomography (EBCT)
The Journal of Heart and Lung Transplantation Volume 18, Number 1
proliferatiion, media degeneration and inflammatory thickening of the adventitia Similar tindiigs were found in allografts tmusfected with P-Gal OT Ad-null adenovhua. ln contrast, lntima thickening was significantly decreased in sFas txansfected grafts (39.20+ 5.29um vs. Allografl 65.60+6.71um, P= 0.004). With sFas, the elastic lamina and thickness of media (71.02+ 4.72urn ys. Allograft 51.34% 2.76um, P= 0.0002) were well preserved compared to other r&grafts and similar to isogratls. Conclusions: Soluble-Fas gene transfer in viva markedly inhibits and delays AGA.
53
OVEREXPREWON INHlBlTS CELL PROlJFERATlON AND P2’ ENHANCES CYCLOSPORINE’S ANTIPROLIFERATIVE EFFECTS: A NOVEL STRATEGY FOR IMMUNOSUPPRESSION. A.K. Khanna, M. Plummer, J.D. Hosenpud. The Medical College of Wisconsin, Milwaukee, WI. The events of cell-cycle control play a crucial role in the cascade of events leading to the allo immune activation in the organ transplant recipient. A number of studies have demonstrated that during T cell activation a number of cyclins and cyclin dependent kinases are activated. It is conceivable that by directly altering the activity of cell cycle-dependent molecules, a more direct approach to immunosuppression can be undenaken without the inherent side effects associated with conventional agents. Using the information that we and others have generated regarding the effects of cyclosporine on TGF-P (an immunosuppressive cytokine) and the effects of TGF-P on the cell cyclin inhibitor ~21, we hypothesize that increasing ~21 levels directly could be used as an immunosuppressive strategy. Using molecularly engineered eukaryotic expression vector plasmids expressing p2 I gene in the sense direction, p2 I gene was over-expressed in vitro in A-549 cells and In-viva in mice. RT-PCR and western blot analysis was used to quantify the expression of p2l in A-S49 cells. Reduced proliferation measured by ‘H-thymidine uptake assay was used as a surrogate for immunosuppression. DBAR mice were injected with 100 pg sense plasmid DNA complexed with lipofectamine. p21 mRNA expression in splenocytes and liver was determined by RT-PCR and proliferation of resting and anti-CD3 stimulated splenocytes were determined as above. Controls were non-injected mice. We were successful in increasing the expression of ~21 mRNA and protein in transfected A-549 cells and mRNA levels in mouse spleen and liver. Cyclosporine inhibits the proliferation of A-549 cells and Imouse splenocytes in a dose dependent fashion. The inhibitory effect (% of control) of 250 rig/ml cyclosporine was augmented in ~21 over-expressing A-S49 cells compared to unmodified cells (4313% vs 15+4%, p
49
and anti-CD3 induced ex-viva proliferation of splenocytes was reduced by 66% (~~0.03) and 70% (p
55
USE OF SEQUENCE SPECIFIC OLIGODEOXYNUCLEOTIDES TO INHIBIT MY-OINTIMAL PROLIFBR4TION ASSOCIATED WITH GRAFT CORONARY ARTERY DISEASE M.P. Ennen, R.S. Poston, B.T. Fe&y, G. Hoyt, R.C. Robbins. Stanford University. Stanford, CA. Backgmuad: Graft coronary artery disease (GCAD) due to myointimal proliferation is a major cause of morbidity and mortality in the late post-transplant period. We hypothesize that antisense oligodeoxynucleotides (AS-GDN) against PCNA and CDC-Zk, proteins necessary for DNA replication and cell cycle progression, respectively, would inhibit smooth muscle proliferation and reduce. GCAD Methods: PVG rat hearts were harvested and incubated ex viva for 45 min at 4” C and 5 atmospheres. Hearts were continuously perfused (0.5 ccfmin) with SO@l AS-ODN, 80 pM Sense (S-ODN), 80 pM Scrambled (SCR-ODN) or vehicle (PBS). Hearts were heterotopically transplanted into AC1 recipients, followed by 7.5n@g CsA x 10 d. Grafts were explanted at POD 90, sectioned, and stained with H&E. Percentage luminal narrowing was quantified using C-Imaging software (Cranbeny Twp, NJ). Results: Treatment with AS-ODN and Sense-ODN significantly reduced myointimal proliferation compared to SCR-GDN and PBS. No significant differences wefe observed between AS-ODN and S-ODN treatment groups or between SCR-ODN and PBS. Transfection efficiency was 6328% of myocytes determined by FITC labeling. N Treatment Group Mean % Cellularity inside Internal Elastic Lamina 15 40.6+12.2 PBS AS ODN 16 16.3+1.0’ 16 17.4+2.2* RAS ODN SCR ODN 13 36.1+11.8 ‘P-zO.001 vs. SCR-ODN and PBS: Student T-Test **P
54
Abstracts
THE IMPACT OF EXPLOSIVE BRAIN DEATH ON THE GENESIS OF CARDIAC ALLOGRAFT VASCULOPATHY: AN INTRAVASCULAR ULTRASOUND STUDY Mandeep R. Mehra, Ananth Prasad, Patricia A. Uber; Myung Park, Robert Scott, Ochsner Medical Institutions, New Orleans, LA. Explosive brain death is accompanied by a sudden increase in intracranial pressure which results in up-regulation of MHC class I and II antigens and CO-stbnulatory molecules, thus contributing to enhanced immunogenic@ of the transplanted heart and susceptibility to post transplant immunologic and non-immunologic insults. Whether these early events influence the later development of cardiac allograft vasculopathy remains unknown. We examined a consecutive cohort of 61 heart transplant recipients between 1993-1995 who undenvent yearly intravascular ultrasound and sought to determine the association of explosive (gun shot wound, closed head trauma, bleed) brain death (BD) and the development of intbnal thickening (IT) as well as cardiac events (sudden death, myocardial infarction, revascularization). Data on the mode of brain death and detailed immunologic and non-immunologic variables were examined co-variate and in a proportional hazards model. Results: Group n Post Transplant Suhival Mean tiximal IT Cardiac (monthr) E”CIm (mm) Explosive BD 27 63+ t9 0.59f 0.1 10 (37%) Gradual
BD
34
72 f t7’
0.32 f 0.29;’
4 (LZ%o)#
*p=oos ‘.p=O.O2 #p=O.Ol In a multivariate model, ntode of BD (1=0.28, p=O.O2) persisted as an independent variable in concert with advanced donor age (1=0.42, p=O.OOOS) and lower average cyclosporine dose (I= - 0.25, p=O.O5). Actuarial survival analysis confmed the significant predictive impact of severe IT (logrank (Mantel-Cox) p=O.O2) and explosive brain death (logrank (Mantel-Cox) p=O.Ol) on the development of cardiac events. Conclusions. Explosive Brain Death is associated with the development of a greater degree of prognostically relevant intimal proliferation afier heart transplantation and is predictive of predilection to cardiac events. These data allude to the seminal importance of pre-transplant donor events in preparing the transplanted vasculature for pathologic events that influence the genesis of cardiac allograft vasculopathy.
56
NONINVASIVE. COST-EFFECTIVE DETECTION OF CARDIAC ALLOGRAFI VASCULOPATHY BY ELECTRON BEAM COMPUTED TOMOGRAPHY (EBCT) M. Hunuuel. F.D Knolhnann*. S. Spiegelsberger. W. Boksch. E. Wellnhofer. R. Felix*. R. Hclzer. Deutsches Herzzenlnun Berlin. Berlin Gernmn~. and *CharitC. Campus Vircho~v-Klisiku111. Berlin. Germany. Purpose: Cardiac allografl vascnlopathy is the most impollant cause of death aner cardiac Iransplantalion once the inunediale posloperative pexiod has been passed. The actually used most senshive melhods for diagnosis are coronan angiography combined with inlracoronary ulrrasound (ICUS). which are both invasive and expensive. Our aim nas lo determine the acwracv of the nouinvasive electron beam compwed tomography (EBCT) (0 identify s;gniIicant coronary artev disease in heart Iransplan( recipienls. Methuds: We examined 50 hear1 transplanl recipienls (44 male and 6 female. age: 32-63 years) 13 - 200 months aller HTs by EBCT lo detect corona0 artery calcifications. Calcilications were quanliIied bx lhe Aga:alston-scoring ~sten~. All subjew underuent cardiac angiography on the same day. and 20 were studied addhionally by intracoronac ullrasound (ICUS). The severi& of Tsw~asculopathy. delennined by angiograpy was graded dependent on lhe degree of epicardial coronary slenosis (> 50%) and by ICUS accorchng to the SIanford classilication. The EBCT calcium score was compared n ilh lhe results of coronas nngiography and of ICUS bx analysis of variance (ANOVA) Results: All but seven patients had a calcium score of greater lhau 0 None of the patien& with a score lowr Iban 55 dwzlosed Ilenlod~nasdcall~ rele\aat stenoses ( > 50%) of the corona0 arteries wualized by angiography. but 8 of 12 patients with a higher score had sigiuficnnl stenoses (pC0 0001). Using this threshold. EBCT had a sensili~ity of 100% and speciticity of YO% ICUS conIirmed the presence of calciIied plaques in all palienls with a score above 50 The EBCT calciunl score of patients wilh Slnnford IV lesions was lO8*32 compared lo only 7-t) in patlen& with Stanford I-III lesions (p
proliferatiion, media degeneration and inflammatory thickening of the adventitia Similar tindiigs were found in allografts tmusfected with P-Gal OT Ad-null adenovhua. ln contrast, lntima thickening was significantly decreased in sFas txansfected grafts (39.20+ 5.29um vs. Allografl 65.60+6.71um, P= 0.004). With sFas, the elastic lamina and thickness of media (71.02+ 4.72urn ys. Allograft 51.34% 2.76um, P= 0.0002) were well preserved compared to other r&grafts and similar to isogratls. Conclusions: Soluble-Fas gene transfer in viva markedly inhibits and delays AGA.
53
OVEREXPREWON INHlBlTS CELL PROlJFERATlON AND P2’ ENHANCES CYCLOSPORINE’S ANTIPROLIFERATIVE EFFECTS: A NOVEL STRATEGY FOR IMMUNOSUPPRESSION. A.K. Khanna, M. Plummer, J.D. Hosenpud. The Medical College of Wisconsin, Milwaukee, WI. The events of cell-cycle control play a crucial role in the cascade of events leading to the allo immune activation in the organ transplant recipient. A number of studies have demonstrated that during T cell activation a number of cyclins and cyclin dependent kinases are activated. It is conceivable that by directly altering the activity of cell cycle-dependent molecules, a more direct approach to immunosuppression can be undenaken without the inherent side effects associated with conventional agents. Using the information that we and others have generated regarding the effects of cyclosporine on TGF-P (an immunosuppressive cytokine) and the effects of TGF-P on the cell cyclin inhibitor ~21, we hypothesize that increasing ~21 levels directly could be used as an immunosuppressive strategy. Using molecularly engineered eukaryotic expression vector plasmids expressing p2 I gene in the sense direction, p2 I gene was over-expressed in vitro in A-549 cells and In-viva in mice. RT-PCR and western blot analysis was used to quantify the expression of p2l in A-S49 cells. Reduced proliferation measured by ‘H-thymidine uptake assay was used as a surrogate for immunosuppression. DBAR mice were injected with 100 pg sense plasmid DNA complexed with lipofectamine. p21 mRNA expression in splenocytes and liver was determined by RT-PCR and proliferation of resting and anti-CD3 stimulated splenocytes were determined as above. Controls were non-injected mice. We were successful in increasing the expression of ~21 mRNA and protein in transfected A-549 cells and mRNA levels in mouse spleen and liver. Cyclosporine inhibits the proliferation of A-549 cells and Imouse splenocytes in a dose dependent fashion. The inhibitory effect (% of control) of 250 rig/ml cyclosporine was augmented in ~21 over-expressing A-S49 cells compared to unmodified cells (4313% vs 15+4%, p
49
and anti-CD3 induced ex-viva proliferation of splenocytes was reduced by 66% (~~0.03) and 70% (p
55
USE OF SEQUENCE SPECIFIC OLIGODEOXYNUCLEOTIDES TO INHIBIT MY-OINTIMAL PROLIFBR4TION ASSOCIATED WITH GRAFT CORONARY ARTERY DISEASE M.P. Ennen, R.S. Poston, B.T. Fe&y, G. Hoyt, R.C. Robbins. Stanford University. Stanford, CA. Backgmuad: Graft coronary artery disease (GCAD) due to myointimal proliferation is a major cause of morbidity and mortality in the late post-transplant period. We hypothesize that antisense oligodeoxynucleotides (AS-GDN) against PCNA and CDC-Zk, proteins necessary for DNA replication and cell cycle progression, respectively, would inhibit smooth muscle proliferation and reduce. GCAD Methods: PVG rat hearts were harvested and incubated ex viva for 45 min at 4” C and 5 atmospheres. Hearts were continuously perfused (0.5 ccfmin) with SO@l AS-ODN, 80 pM Sense (S-ODN), 80 pM Scrambled (SCR-ODN) or vehicle (PBS). Hearts were heterotopically transplanted into AC1 recipients, followed by 7.5n@g CsA x 10 d. Grafts were explanted at POD 90, sectioned, and stained with H&E. Percentage luminal narrowing was quantified using C-Imaging software (Cranbeny Twp, NJ). Results: Treatment with AS-ODN and Sense-ODN significantly reduced myointimal proliferation compared to SCR-GDN and PBS. No significant differences wefe observed between AS-ODN and S-ODN treatment groups or between SCR-ODN and PBS. Transfection efficiency was 6328% of myocytes determined by FITC labeling. N Treatment Group Mean % Cellularity inside Internal Elastic Lamina 15 40.6+12.2 PBS AS ODN 16 16.3+1.0’ 16 17.4+2.2* RAS ODN SCR ODN 13 36.1+11.8 ‘P-zO.001 vs. SCR-ODN and PBS: Student T-Test **P
54
Abstracts
THE IMPACT OF EXPLOSIVE BRAIN DEATH ON THE GENESIS OF CARDIAC ALLOGRAFT VASCULOPATHY: AN INTRAVASCULAR ULTRASOUND STUDY Mandeep R. Mehra, Ananth Prasad, Patricia A. Uber; Myung Park, Robert Scott, Ochsner Medical Institutions, New Orleans, LA. Explosive brain death is accompanied by a sudden increase in intracranial pressure which results in up-regulation of MHC class I and II antigens and CO-stbnulatory molecules, thus contributing to enhanced immunogenic@ of the transplanted heart and susceptibility to post transplant immunologic and non-immunologic insults. Whether these early events influence the later development of cardiac allograft vasculopathy remains unknown. We examined a consecutive cohort of 61 heart transplant recipients between 1993-1995 who undenvent yearly intravascular ultrasound and sought to determine the association of explosive (gun shot wound, closed head trauma, bleed) brain death (BD) and the development of intbnal thickening (IT) as well as cardiac events (sudden death, myocardial infarction, revascularization). Data on the mode of brain death and detailed immunologic and non-immunologic variables were examined co-variate and in a proportional hazards model. Results: Group n Post Transplant Suhival Mean tiximal IT Cardiac (monthr) E”CIm (mm) Explosive BD 27 63+ t9 0.59f 0.1 10 (37%) Gradual
BD
34
72 f t7’
0.32 f 0.29;’
4 (LZ%o)#
*p=oos ‘.p=O.O2 #p=O.Ol In a multivariate model, ntode of BD (1=0.28, p=O.O2) persisted as an independent variable in concert with advanced donor age (1=0.42, p=O.OOOS) and lower average cyclosporine dose (I= - 0.25, p=O.O5). Actuarial survival analysis confmed the significant predictive impact of severe IT (logrank (Mantel-Cox) p=O.O2) and explosive brain death (logrank (Mantel-Cox) p=O.Ol) on the development of cardiac events. Conclusions. Explosive Brain Death is associated with the development of a greater degree of prognostically relevant intimal proliferation afier heart transplantation and is predictive of predilection to cardiac events. These data allude to the seminal importance of pre-transplant donor events in preparing the transplanted vasculature for pathologic events that influence the genesis of cardiac allograft vasculopathy.
56
NONINVASIVE. COST-EFFECTIVE DETECTION OF CARDIAC ALLOGRAFI VASCULOPATHY BY ELECTRON BEAM COMPUTED TOMOGRAPHY (EBCT) M. Hunuuel. F.D Knolhnann*. S. Spiegelsberger. W. Boksch. E. Wellnhofer. R. Felix*. R. Hclzer. Deutsches Herzzenlnun Berlin. Berlin Gernmn~. and *CharitC. Campus Vircho~v-Klisiku111. Berlin. Germany. Purpose: Cardiac allografl vascnlopathy is the most impollant cause of death aner cardiac Iransplantalion once the inunediale posloperative pexiod has been passed. The actually used most senshive melhods for diagnosis are coronan angiography combined with inlracoronary ulrrasound (ICUS). which are both invasive and expensive. Our aim nas lo determine the acwracv of the nouinvasive electron beam compwed tomography (EBCT) (0 identify s;gniIicant coronary artev disease in heart Iransplan( recipienls. Methuds: We examined 50 hear1 transplanl recipienls (44 male and 6 female. age: 32-63 years) 13 - 200 months aller HTs by EBCT lo detect corona0 artery calcifications. Calcilications were quanliIied bx lhe Aga:alston-scoring ~sten~. All subjew underuent cardiac angiography on the same day. and 20 were studied addhionally by intracoronac ullrasound (ICUS). The severi& of Tsw~asculopathy. delennined by angiograpy was graded dependent on lhe degree of epicardial coronary slenosis (> 50%) and by ICUS accorchng to the SIanford classilication. The EBCT calcium score was compared n ilh lhe results of coronas nngiography and of ICUS bx analysis of variance (ANOVA) Results: All but seven patients had a calcium score of greater lhau 0 None of the patien& with a score lowr Iban 55 dwzlosed Ilenlod~nasdcall~ rele\aat stenoses ( > 50%) of the corona0 arteries wualized by angiography. but 8 of 12 patients with a higher score had sigiuficnnl stenoses (pC0 0001). Using this threshold. EBCT had a sensili~ity of 100% and speciticity of YO% ICUS conIirmed the presence of calciIied plaques in all palienls with a score above 50 The EBCT calciunl score of patients wilh Slnnford IV lesions was lO8*32 compared lo only 7-t) in patlen& with Stanford I-III lesions (p