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Apoptosis and expression of heme oxygenase-1 in heart transplant recipients during acute rejection episodes
Apoptosis and Expression of Heme Oxygenase-1 in Heart Transplant Recipients During Acute Rejection Episodes M.K. Chok, M. Se´ne´chal, R. Dorent, Z. Mallat, P. Leprince, N. Bonnet, A. Pavie, J.J. Ghossoub, and I. Gandjbakhch
A
LTHOUGH the success of heart transplantation procedures has improved during the last few years, acute rejection (AR) remains a leading cause of morbidity and mortality. The importance of apoptosis during AR episodes is not completely understood. Programmed cell death is a dynamic phenomenon regulated by stimulating and inhibiting proteins that can be initiated by intrinsic signals, or by extrinsic signals, such as cytokines, hormones, oxidized lipids, or viral agents.1 The balance between anti-apoptotic and pro-apoptotic proteins is critical to determining whether a cell undergoes apoptosis. During acute and chronic rejection, apoptosis has been shown in heart transplant animals and humans.2– 4 Apoptotic nuclei have been observed in myocytes, endothelial cells, and activated host monocytes/macrophages. Expression of heme oxygenase-1 (HO-1) seems to suppress rejection in a mouse-to-rat cardiac transplant model.5 The present study was conducted to verify the relation between acute rejection and apoptosis; apoptosis and the expression of HO-1, and the expression of HO-1 and AR. MATERIALS AND METHODS This study includes 18 heart transplant patients operated between December 1998 and December 1999 (15 men, 3 women; mean age, 51 years). Mean donor age was 38 years. All patients received rabbit anti-thymocyte globulin as induction therapy. Cyclosporine, prednisone, and azathioprine were administered for maintenance immunosuppression. Cyclosporine was initiated after the operation with doses adjusted according to renal function. In nine patients the biopsy was performed the first week after heart transplant in the absence of rejection. The other nine patients had a biopsy selected during an AR episode. AR diagnosis and grading was made with light microscopy according to the criteria standardized by the International Society of Heart and Lung Transplantation.
Tissue Preparation The tissue fragments were obtained from the right interventricular septum with a biotome introduced through the right internal jugular vein. The tissue fragments were fixed in 4% buffered formaldehyde and dried with PBS sucrose 30%. The tissue fragments were conserved at ⫺80°C. For each biopsy, eight consequtive sections were mounted on eight separate slides. This study used only three to four consequtives slides for distinct marking by TUNEL or HO-1.
Morphology Analysis of Apoptosis With TUNEL Method The TUNEL method is used to visualize DNA cleavage. The method is based on the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling at sites of DNA breaks. After hydration and inactivation of endogen peroxydase with oxygenated water, we covered the slides with TdT (5cc) and bioUTP (45 cc) and incubated for 60 minutes at 37°C. After carefully wiping the excess fluid, we incubated the slides in the presence of avidine peroxydase complex (Victastain ABC kit PK-6100, Vector SA r). The slides were rinsed and incubated for 20 minutes in the presence of chromogen 3-amino-9-ethylcarberol.
Detection of HO-1 Expression The detection of HO-1 expression was realized with immunized rabbit serum. The slides were incubated with a solution of PBSBSA 2% for 20 minutes and then with oxygenated water 3% at room temperature for 15 minutes to block endogeous peroxidase. The slides were incubated with antibodies for 1 hour. The slides were rinsed with PBS and then incubated with antibodies directed against the first antibodies biotiylated for 1 hour at room temperature. After rinsing with PBS, 100 cc of Victastain ABC Elite PK-6100 (vector SA) were put for 30 minutes on the slides. After rinsing, the activity of peroxidase is visualized with diaminobenzidine as the chromogen. The level of expression was estimated by a semiquantitative scale for the optic intensity of cells: 0 ⫽ no marking, 1 ⫽ very weak marking, 2 ⫽ weak marking, 3 ⫽ moderate marking, and 4 ⫽ intense marking. Two groups of biopsies were analyzed. Group GO (n ⫽ 9), representing biopsies without rejection. Group GR (n ⫽ 9) representing biopsy with rejection (six biopsies had a rejection grade of 1A and three patients had a rejection grade of 1B). Results concerning HO-1 expression and apoptosis were compared with Student’s t test; logistic regression was carried out for the correlation between apoptosis and HO-1 expression. In all tests used, the significance level was defined as P ⬍. 05. From the Service de chirurgie Cardio-Vasculaire et Thoracique, Groupe Hospitalier Pitie´-Salpeˆtrie`re, (M.K.C., M.S., R.D., P.L., N.B., A.P, J.J.G, I.G.), and Inserm, U 541 Dynamique Cardiocirculatoire, Biologie de la Paroi Vasculaire (Z.M.), Paris, France. Address reprint requests to Richard Dorent, MD, Service de Chirurgie Cardio-vasculaire et Thoracique, Groupe Hospitalier Pitie´-Salpeˆtrie`re, 83, boulevard de l’Hoˆpital, 75013 Paris, France. E-mail: [email protected].
© 2002 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/02/$–see front matter PII S0041-1345(02)03666-7
Transplantation Proceedings, 34, 3239 –3240 (2002)
3239
CHOK, SE´ NE´ CHAL, DORENT ET AL
3240
RESULTS
DISCUSSION
For each section, we identified an average of 579 myocytes, 469 graft-infiltrating cells, and 6 vessels.
In this study we demonstrated that during rejection episodes of grade ⬍2 the apoptotic index is elevated in endothelial cells and infiltrating cells in heart transplants. Expression of HO-1 was reduced during AR episodes in endothelial cells, infiltrating cells, and cardiac myocytes. There was also an inverse correlation between the expression of HO-1 and the degree of apoptosis in cardiac myocytes, infiltrating graft cells, and endothelial cells. In a previous study, expression of HO-1 was observed to correlate with cardiac xenograft survival.6 In a mouse-to-rat cardiac transplant model, HO-1 expression was required to prevent vascular thrombosis and suppress xenograft infiltration by activated host leukocytes.5 The anti-apoptotic properties of HO-1 are thought to rely on the ability of this enzyme to degrade heme and generate bilirubin, free iron, and carbon monoxide. Bilirubin is a potent anti-oxidant, free iron upregulates the transcription of the cytoprotective gene, and carbon monoxide is thought to be essential in regulating vascular relaxation in a manner similar to nitric oxide.7,8 Also, CO suppresses graft rejection by inhibiting platelet aggregation that facilitates vascular thrombosis and myocardial infarction.5 In conclusion, apoptosis of cardiac myocytes, graft infiltrating cells, and endothelial cells is one of the mechanisms of immune-mediated death during AR. Expression of heme oxygenase is less during episodes of acute rejection. This finding suggests that the expression of this cytoprotective antigen may prevent apoptosis and presumably thereby may contribute to sustain graft function.
Detection of Apoptosis by TUNEL Method and Its Relationship With Acute Rejection
Cardiac Myocytes Global apoptotic index of cardiac myocytes was 0.35 ⫾ 0.04%. Apoptotic index of group GO (0.23 ⫾ 0.03%), and group GR (0.32 ⫾ 0.05%) were comparable. Graft Infiltrating Cells Cells were abundant in biopsy done during the first week posttransplantation and during an AR episode. The majority of graft infiltrating cells were localized to the periphery of microvessels. The global apoptotic index of those cells was 4.13 ⫾ 0.21%. The apoptotic index of the group GO (2.94 ⫾ 0.29%) was significantly lower than that of group GR (4.89 ⫾ 0.42%, P ⬍ .0001). Endothelial Cells Apoptosis of endothelial cells was evaluated at the level of myocardial micro vessels. Global apoptotic index was 44.46 ⫾ 0.19. The apoptotic index of group GO (33.12 ⫾ 2.98%) was significantly lower than group GR (50.65 ⫾ 3.45%, P ⬍ .0001). Expression of HO-1 and AR Expression of HO-1 was more significant in cardiac myocytes (2.37 ⫾ 0.08). Expression of HO-1 was less in endothelial cells (1.48 ⫾ 0.06) and in grafts infiltrating cells (0.97 ⫾ 0.05). Cardiac Myocytes Expression of HO-1 (2.59 ⫾ 0.13 versus 2.40 ⫾ 0.14) between group GO and GR was not different. Graft Infiltrating Cells Expression of HO-1 was significantly lower in group GR (0.89 ⫾ 0.07) than group GO (1.23 ⫾ 0.08, P ⫽ .042). Endothelial Cells Expression of HO-1 was significantly lower in group GR (1.32 ⫾ 0.10, P ⬍ .05) than group GO (1.67 ⫾ 0.10). Correlation Between Apoptosis and Expression of HO-1 For each type of cells we found an inverse correlation between expression of HO-1 and apoptotic index. The correlation was more pronounced in cardiac myocytes (r ⫽ 0.50) than infiltrating cells (r ⫽ 0.45) and endothelial cells (r ⫽ 0.41).
REFERENCES 1. Mallat Z, Tedgui A: Br J Pharma 130:947, 2000 2. Kageyama Y, Li XK, Suzuki S, et al: Ann Thorac Surg 65:1604, 1998 3. Laguens RP, Merckert PMC, San Martino J, et al: J Heart Lung Transplant 15:911, 1996 4. Xu B, Sakkas LI, Slachta CA, et al: Transplantation 71:1137, 2001 5. Sato K, Balla J, Otterbein L, et al: J Immunol 166:4185, 2001 6. Soares MP, Lin Y, Anrather J, et al: Nature Med 4:1073, 1998 7. Szaboles MJ, Michler RE, Yang X, et al: Circulation 94:1665, 1996 8. Szaboles MJ, Ravalli S, Minanov O, et al: Transplantation 65:804, 1998
A
LTHOUGH the success of heart transplantation procedures has improved during the last few years, acute rejection (AR) remains a leading cause of morbidity and mortality. The importance of apoptosis during AR episodes is not completely understood. Programmed cell death is a dynamic phenomenon regulated by stimulating and inhibiting proteins that can be initiated by intrinsic signals, or by extrinsic signals, such as cytokines, hormones, oxidized lipids, or viral agents.1 The balance between anti-apoptotic and pro-apoptotic proteins is critical to determining whether a cell undergoes apoptosis. During acute and chronic rejection, apoptosis has been shown in heart transplant animals and humans.2– 4 Apoptotic nuclei have been observed in myocytes, endothelial cells, and activated host monocytes/macrophages. Expression of heme oxygenase-1 (HO-1) seems to suppress rejection in a mouse-to-rat cardiac transplant model.5 The present study was conducted to verify the relation between acute rejection and apoptosis; apoptosis and the expression of HO-1, and the expression of HO-1 and AR. MATERIALS AND METHODS This study includes 18 heart transplant patients operated between December 1998 and December 1999 (15 men, 3 women; mean age, 51 years). Mean donor age was 38 years. All patients received rabbit anti-thymocyte globulin as induction therapy. Cyclosporine, prednisone, and azathioprine were administered for maintenance immunosuppression. Cyclosporine was initiated after the operation with doses adjusted according to renal function. In nine patients the biopsy was performed the first week after heart transplant in the absence of rejection. The other nine patients had a biopsy selected during an AR episode. AR diagnosis and grading was made with light microscopy according to the criteria standardized by the International Society of Heart and Lung Transplantation.
Tissue Preparation The tissue fragments were obtained from the right interventricular septum with a biotome introduced through the right internal jugular vein. The tissue fragments were fixed in 4% buffered formaldehyde and dried with PBS sucrose 30%. The tissue fragments were conserved at ⫺80°C. For each biopsy, eight consequtive sections were mounted on eight separate slides. This study used only three to four consequtives slides for distinct marking by TUNEL or HO-1.
Morphology Analysis of Apoptosis With TUNEL Method The TUNEL method is used to visualize DNA cleavage. The method is based on the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling at sites of DNA breaks. After hydration and inactivation of endogen peroxydase with oxygenated water, we covered the slides with TdT (5cc) and bioUTP (45 cc) and incubated for 60 minutes at 37°C. After carefully wiping the excess fluid, we incubated the slides in the presence of avidine peroxydase complex (Victastain ABC kit PK-6100, Vector SA r). The slides were rinsed and incubated for 20 minutes in the presence of chromogen 3-amino-9-ethylcarberol.
Detection of HO-1 Expression The detection of HO-1 expression was realized with immunized rabbit serum. The slides were incubated with a solution of PBSBSA 2% for 20 minutes and then with oxygenated water 3% at room temperature for 15 minutes to block endogeous peroxidase. The slides were incubated with antibodies for 1 hour. The slides were rinsed with PBS and then incubated with antibodies directed against the first antibodies biotiylated for 1 hour at room temperature. After rinsing with PBS, 100 cc of Victastain ABC Elite PK-6100 (vector SA) were put for 30 minutes on the slides. After rinsing, the activity of peroxidase is visualized with diaminobenzidine as the chromogen. The level of expression was estimated by a semiquantitative scale for the optic intensity of cells: 0 ⫽ no marking, 1 ⫽ very weak marking, 2 ⫽ weak marking, 3 ⫽ moderate marking, and 4 ⫽ intense marking. Two groups of biopsies were analyzed. Group GO (n ⫽ 9), representing biopsies without rejection. Group GR (n ⫽ 9) representing biopsy with rejection (six biopsies had a rejection grade of 1A and three patients had a rejection grade of 1B). Results concerning HO-1 expression and apoptosis were compared with Student’s t test; logistic regression was carried out for the correlation between apoptosis and HO-1 expression. In all tests used, the significance level was defined as P ⬍. 05. From the Service de chirurgie Cardio-Vasculaire et Thoracique, Groupe Hospitalier Pitie´-Salpeˆtrie`re, (M.K.C., M.S., R.D., P.L., N.B., A.P, J.J.G, I.G.), and Inserm, U 541 Dynamique Cardiocirculatoire, Biologie de la Paroi Vasculaire (Z.M.), Paris, France. Address reprint requests to Richard Dorent, MD, Service de Chirurgie Cardio-vasculaire et Thoracique, Groupe Hospitalier Pitie´-Salpeˆtrie`re, 83, boulevard de l’Hoˆpital, 75013 Paris, France. E-mail: [email protected].
© 2002 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/02/$–see front matter PII S0041-1345(02)03666-7
Transplantation Proceedings, 34, 3239 –3240 (2002)
3239
CHOK, SE´ NE´ CHAL, DORENT ET AL
3240
RESULTS
DISCUSSION
For each section, we identified an average of 579 myocytes, 469 graft-infiltrating cells, and 6 vessels.
In this study we demonstrated that during rejection episodes of grade ⬍2 the apoptotic index is elevated in endothelial cells and infiltrating cells in heart transplants. Expression of HO-1 was reduced during AR episodes in endothelial cells, infiltrating cells, and cardiac myocytes. There was also an inverse correlation between the expression of HO-1 and the degree of apoptosis in cardiac myocytes, infiltrating graft cells, and endothelial cells. In a previous study, expression of HO-1 was observed to correlate with cardiac xenograft survival.6 In a mouse-to-rat cardiac transplant model, HO-1 expression was required to prevent vascular thrombosis and suppress xenograft infiltration by activated host leukocytes.5 The anti-apoptotic properties of HO-1 are thought to rely on the ability of this enzyme to degrade heme and generate bilirubin, free iron, and carbon monoxide. Bilirubin is a potent anti-oxidant, free iron upregulates the transcription of the cytoprotective gene, and carbon monoxide is thought to be essential in regulating vascular relaxation in a manner similar to nitric oxide.7,8 Also, CO suppresses graft rejection by inhibiting platelet aggregation that facilitates vascular thrombosis and myocardial infarction.5 In conclusion, apoptosis of cardiac myocytes, graft infiltrating cells, and endothelial cells is one of the mechanisms of immune-mediated death during AR. Expression of heme oxygenase is less during episodes of acute rejection. This finding suggests that the expression of this cytoprotective antigen may prevent apoptosis and presumably thereby may contribute to sustain graft function.
Detection of Apoptosis by TUNEL Method and Its Relationship With Acute Rejection
Cardiac Myocytes Global apoptotic index of cardiac myocytes was 0.35 ⫾ 0.04%. Apoptotic index of group GO (0.23 ⫾ 0.03%), and group GR (0.32 ⫾ 0.05%) were comparable. Graft Infiltrating Cells Cells were abundant in biopsy done during the first week posttransplantation and during an AR episode. The majority of graft infiltrating cells were localized to the periphery of microvessels. The global apoptotic index of those cells was 4.13 ⫾ 0.21%. The apoptotic index of the group GO (2.94 ⫾ 0.29%) was significantly lower than that of group GR (4.89 ⫾ 0.42%, P ⬍ .0001). Endothelial Cells Apoptosis of endothelial cells was evaluated at the level of myocardial micro vessels. Global apoptotic index was 44.46 ⫾ 0.19. The apoptotic index of group GO (33.12 ⫾ 2.98%) was significantly lower than group GR (50.65 ⫾ 3.45%, P ⬍ .0001). Expression of HO-1 and AR Expression of HO-1 was more significant in cardiac myocytes (2.37 ⫾ 0.08). Expression of HO-1 was less in endothelial cells (1.48 ⫾ 0.06) and in grafts infiltrating cells (0.97 ⫾ 0.05). Cardiac Myocytes Expression of HO-1 (2.59 ⫾ 0.13 versus 2.40 ⫾ 0.14) between group GO and GR was not different. Graft Infiltrating Cells Expression of HO-1 was significantly lower in group GR (0.89 ⫾ 0.07) than group GO (1.23 ⫾ 0.08, P ⫽ .042). Endothelial Cells Expression of HO-1 was significantly lower in group GR (1.32 ⫾ 0.10, P ⬍ .05) than group GO (1.67 ⫾ 0.10). Correlation Between Apoptosis and Expression of HO-1 For each type of cells we found an inverse correlation between expression of HO-1 and apoptotic index. The correlation was more pronounced in cardiac myocytes (r ⫽ 0.50) than infiltrating cells (r ⫽ 0.45) and endothelial cells (r ⫽ 0.41).
REFERENCES 1. Mallat Z, Tedgui A: Br J Pharma 130:947, 2000 2. Kageyama Y, Li XK, Suzuki S, et al: Ann Thorac Surg 65:1604, 1998 3. Laguens RP, Merckert PMC, San Martino J, et al: J Heart Lung Transplant 15:911, 1996 4. Xu B, Sakkas LI, Slachta CA, et al: Transplantation 71:1137, 2001 5. Sato K, Balla J, Otterbein L, et al: J Immunol 166:4185, 2001 6. Soares MP, Lin Y, Anrather J, et al: Nature Med 4:1073, 1998 7. Szaboles MJ, Michler RE, Yang X, et al: Circulation 94:1665, 1996 8. Szaboles MJ, Ravalli S, Minanov O, et al: Transplantation 65:804, 1998