- Email: [email protected]
Endothelial Injury, an Intriguing Effect of Methotrexate and Cyclophosphamide During Hematopoietic Stem Cell Transplantation in Mice
Endothelial Injury, an Intriguing Effect of Methotrexate and Cyclophosphamide During Hematopoietic Stem Cell Transplantation in Mice L. Zeng, Z. Yan, S. Ding, K. Xu, and L. Wang ABSTRACT Objective. Elevated circulating endothelial cells (EC) in peripheral blood are an important indicator of endothelial damage and graft-versus-host disease (GVHD). However, the injured endothelial vasculature may in turn promote GVHD. In this study, we investigated whether methotrexate or cyclophosphamide, two conventional chemotherapeutic agents used in hematopoietic stem cell transplantation, caused endothelial injury and what were the functional consequences of this injury on GVHD. Methods. Six to 8-week-old female mice were randomly separated into three groups, including methotrexate (15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, and 11), cyclophosphamide (60 mg/kg; days 1, 2), and PBS saline alone (control). Circulating EC (CD31⫹CD133⫺CD45low) and the CD4⫹/CD8⫹ T lymphocytes in the peripheral blood were estimated by flow cytometry on days 1, 3, 5, 7, 9, 11, 16, 21, and 26. The morphologic changes of the endothelium were examined by phase contrast light microscopy to determine the integrity of the endothelial vasculature. Results. Elevated EC were detected at day 1 or 3 in mice receiving cyclophosphamide or methotrexate, respectively, with a peak increase at day 5 or day 3, respectively. The ratio of CD4⫹/CD8⫹ T lymphocytes showed a delayed increase in the peak to day 11 for both groups. In the cyclophosphamide group, there was significant apomorphosis with necrosis/ thrombosis under light microscopy, whereas only apomorphosis was noticed in the methotrexate group. In both groups, EC showed hydropsia and cytomembrane damage. Conclusions. Circulating EC increase during the early phases of cyclophosphamide or methotrexate conditioning, suggesting that both chemotherapeutic drugs induce endothelial damage, which occurs a little earlier than suppression of the immune system.
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ASCULAR ENDOTHELIUM represents a barrier between circulating blood cells and the subendothelial matrix.1 Endothelial integrity has the potential to prevent platelet aggregation and activation, and to inhibit the activity of thrombin or other blood coagulation factors, thereby maintaining blood circulation.2 Endothelial cells (EC) regulate vasodilatation/vasoconstriction by release of nitric oxide, von Willibrandfactor, and endothelin-1 (ET-1), which are involved in immunoreactions, inflammation, and thrombopoiesis.3 Hemopoietic stem cell transplantation (HSCT), which includes using chemotherapeutic agents and irradiation to transplant stem cells, may induce endothelial cell damage,4,5 cause blood coagulation and thrombogenesis, and
affect organ function. Meanwhile, the injured endothelial vasculature in turn may promotes graft-versus-host disease (GVHD). In previous reports pretreatment or GVHD have been shown to cause endothelial injury,6 although the single From the Department of Hematology, Xuzhou Medical College, Xuzhou, P.R. China. Supported by the fund of national nature science fundation (30770915) of P.R. China, the fund of Jiangsu nature science fundation (BK2007504), and the lingjunrencai project, Jiangsu Ministery of Health Foundation, P.R. China. Address reprint requests to Kailin Xu, Department of Hematology, Affiliated Hospital of Xuzhou Medical College, West Huaihai Road 99 (221002), Xuzhou, Jiangsu, PR China. E-mail: [email protected]
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.06.038
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2670
Transplantation Proceedings, 40, 2670 –2673 (2008)
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Fig 1. Effect of methotrexate and cyclophosphamide on endothelial cells and T lymphocytes. A. Methotrexate group. B. Cyclophosphamide group.
damaged factor during HSCT is unknown. In the present study, we examined the role of single damaging factor— conventional chemotherapeutic agents used in HSCT (methotrexate or cyclophosphamide)—in relation to the time point conditioning for a HSCT with respect to endothelial injury and its functional consequences on GVHD. MATERIALS AND METHODS Mice and Reagents Six-week-old female Balb/c mice were purchased from the Animal Center in Xuzhou medical college (Jiangsu). These studies approved by our Institutional Animal Care and Use Committee housed and fed hosts in accordance with our animal center guidelines. PE-Cy5– conjugated rat anti-mouse CD45 monoclonal antibody and flourescein isothiocyanate (FITC)-conjugated rat anti-mouse CD31 monoclonal antibody were purchased from BD Pharmingen. A PE-conjugated rat anti-mouse CD133 monoclonal antibody was purchased from eBioscience.
Treatment With Methotrexate and Cyclophosphamide Three groups of mice were studied as for endothelial injury during bone marrow transplantation: methotrexate group (15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, and 11); cyclophosphamide group (60 mg/kg on days 1 and 2), and phosphate-buffered saline (control). The drugs were administered by intraperitoneal injection.
Monitoring the Circulating Endothelial Cells and CD4⫹/CD8⫹ T Cells Blood samples were obtained from mice on days 1, 3, 5, 7, 9, 11, 16, 21, and 26. Each sample was divided. One was incubated for 20 minutes at 4°C with PE-Cy5– conjugated rat anti-mouse CD45 monoclonal antibody, FITC-conjugated rat anti-mouse CD31 monoclonal antibody, or PE-conjugated rat anti-mouse CD133 monoclonal antibody. The other was incubated for 20 minutes at 4°C with FITC-conjugated rat anti-mouse CD3 and PE-conjugated rat anti-mouse CD4 monoclonal antibody/PE-conjugated rat antimouse CD8 monoclonal antibody monoclonal antibody. The ECs (CD133⫺, CD31⫹, CD45low)7 and CD3⫹CD4⫹/CD3⫹CD8⫹ T lymphocytes were enumerated by fluorescent activated cell sorting.
Tissue Harvest and Fixation Liver, skin, and intestinal tissues were harvested from mice on days 1, 3, 5, 7, and 9. A systemic blood vessel was flushed first with physiologic solution to clear the blood from the tissue, then perfused with fixation solution containing 2.5% glutaraldehyde and
1% paraform. The tissue specimen for ultrastructural or endothelial integrity by transmission electron microscopy was fixed with 2.5% glutaraldehyde, and that for morphology of tissue was fixed in 4% paraformaldehyde.
Assessing Morphologic Changes To assess morphologic changes, 4-m paraffin sections were stained with hematoxylin and eosin for histologic examination. The changes in endothelium and the necrotic tissue were assessed by light microscopy.
Statistical Analysis Data were expressed as mean values ⫾ standard deviations with P ⬍ .05 considered significant.
RESULTS Pristine Elevation of Circulating Endothelial Cells After Conditioning
The numbers of circulating ECs in the methotrexate group or the cyclophosphamide group were elevated abruptly at day 3 and day 1, respectively, gradually decreasing thereafter. Before they decreased, the number of cells in the methotrexate group were high for 13 days (Fig 1). Meanwhile, in both groups there was also a decrease in cells, especially CD8⫹ T cell. The ratio of CD4⫹ and CD8⫹ T cells increased gradually up to a peak, and thereafter recovered to normal levels. The time courses in both groups were different. In the methotrexate group, the ratio of T-lymphocyte subpopulations increased at day 7 and peaked at day 11. In the cyclophosphamide group, the time course was earlier— 4 days (Fig 1). We concluded that the immunosuppressive effects for these drugs occurred later than the endothelium injury. Changes in Morphology
Under light microscopy, the histologic changes occurred 6 days after conditioning. It was obviously different between the two groups. In the cyclophosphamide group, there was significant apomorphosis, necrosis, and thrombosis. In contrast, there were no visible histopathologic changes of ECs, except edema of parenchymal cells in the methotrexate group (Fig 2). DISCUSSION
Conditioning and immunosuppression are the key process in HSCT. However, dysfunction and failure are always the
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ZENG, YAN, DING ET AL
Fig 2. Pathologic changes of liver and cutaneous post conditioning. A. Liver from the methotrexate group. Hepatocytes were extensively hydropic, although the endothelium showed no visible pathologic changes (original magnification ⫻400). B. Liver from the cyclophosphamide group. Severe hydrops, thrombogenesis, and necrosis are present. C. Skin from the Methotrexate group (original magnification ⫻1000) Interstitium and endothelium showed no visible pathologic changes. D. Skin from the cyclophosphamide group (original magnification ⫻400). Severe hemorrhage is present.
challenges during transplantation. Previous documents have shown that excessive chemotherapy did not contribute to graft survival, but that high-dose immunosuppressants are responsible for infection. Methotrexate and cyclophosphamide, two conventional chemotherapeutic agents used in HSCT, depress or relieve rejection and GVHD. Our data showed that the two drug quickly lead to T-lymphocyte depletion, especially CD8⫹ T lymphocytes, which were predominant after conditioning for 1 week. A remarkable discovery was circulating ECs elevated before the change in T-lymphocyte subpopulations. Previous studies8 have reported that conditioning may damage the endothelium using circulating ECs as a marker of endothelial damage in allogeneic HSCT. Using this marker, we monitored both cyclophosphamide- and methotrexate-damaged endothelium before immune cells. This mechanism needs further investigation. It is well known that an integrated endothelium is the precondition of organ function. The endothelial monolayer at the interface between the extravascular space and the blood plays a crucial role in vascular homeostasis.9 The endothelial monolayer helps to maintain the antithrombotic, anti-inflammatory state of the microvascular bed. Additionally, it controls the tone and proliferative state of the underlying vascular smooth muscle cells. The loss of the endothelial barrier function is associated with severe consequences: tissue edema formation, thrombus formation, neointimal thickening, and abnormal responses to endothelium-dependent agonists.10
To understand the origin of increasing ECs in peripheral blood, we detected a change in morphology. There was a more significant EC degeneration with an increasing diastem between ECs and basilemma. Thrombus formation suggested that circulating ECs, at least partly, came from the damaged endothelium. It further confirmed that the number of circulatory ECs may be considered to be an important indicator of endothelial damage. From these data, we also discovered that the elevation of circulating ECs preceded histologic damage, providing an early marker of slight damage, which might be reversed by timely intervention. The reversibility of the endothelial injury reflects its severity as well as the capacity of cells to undergo the repair necessary for reconstitution of the endothelial barrier. Recovery of endothelial integrity after vascular injury is vital for endothelial barrier function and vascular homeostasis. Hence, further investigations are needed to determine whether circulating ECs are a marker of preservation or repair. REFERENCES 1. Biedermann BC: Vascular endothelial cells: an interesting immunological barrier. Schweiz Med Wochenschr 129:1712, 1999 2. Shimoni A, Yeshurun M, Hardan I, et al: Thrombotic microangiopathy after allogeneic stem cell transplantation in the era of reduced-intensity conditioning: the incidence is not reduced. Biol Blood Marrow Transplant 10:484, 2004 3. Potenza MA, Marasciuolo FL, Chieppa DM, et al: Insulin resistance in spontaneously hypertensive rats is associated with endo-
ENDOTHELIAL INJURY thelial dysfunction characterized by imbalance between NO and ET-1 production. Am J Physiol Heart Circ Physiol 289:H813, 2005 4. Paris F, Fuks Z, Kang A, et al: Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. Science 293:293, 2001 5. Ertault-Daneshpouy M, Leboeuf C, Lemann M, et al: Pericapillary hemorrhage as criterion of severe human digestive graftversus-host disease. Blood 5:1548, 2003 6. Ertault-Daneshpouy M, Leboeuf C, Lemann M, et al: Pericapillary hemorrhage as criterion of severe human digestive graftversus-host disease Blood 103:4681, 2004 7. Duda DG, Cohen KS, di Tomaso E, et al: Differential CD146 expression on circulating versus tissue endothelial cells in rectal
2673 cancer patients: implications for circulating endothelial and progenitor cells as biomarkers for antiangiogenic therapy. J Clin Oncol 24:1449, 2006 8. Woywodt A, Scheer J, Hambach L, et al: Circulating endothelial cells as a marker of endothelial damage in allogeneic hematopoietic stem cell transplantation. Blood 103:3603, 2004 9. Cines DB, Pollak ES, Buck CA, et al: Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 91:3527, 1998 10. McFadden EP, Bauters C, Lablanche JM, et al: Response of human coronary arteries to serotonin after injury by coronary angioplasty. Circulation 88:2076, 1993
V
ASCULAR ENDOTHELIUM represents a barrier between circulating blood cells and the subendothelial matrix.1 Endothelial integrity has the potential to prevent platelet aggregation and activation, and to inhibit the activity of thrombin or other blood coagulation factors, thereby maintaining blood circulation.2 Endothelial cells (EC) regulate vasodilatation/vasoconstriction by release of nitric oxide, von Willibrandfactor, and endothelin-1 (ET-1), which are involved in immunoreactions, inflammation, and thrombopoiesis.3 Hemopoietic stem cell transplantation (HSCT), which includes using chemotherapeutic agents and irradiation to transplant stem cells, may induce endothelial cell damage,4,5 cause blood coagulation and thrombogenesis, and
affect organ function. Meanwhile, the injured endothelial vasculature in turn may promotes graft-versus-host disease (GVHD). In previous reports pretreatment or GVHD have been shown to cause endothelial injury,6 although the single From the Department of Hematology, Xuzhou Medical College, Xuzhou, P.R. China. Supported by the fund of national nature science fundation (30770915) of P.R. China, the fund of Jiangsu nature science fundation (BK2007504), and the lingjunrencai project, Jiangsu Ministery of Health Foundation, P.R. China. Address reprint requests to Kailin Xu, Department of Hematology, Affiliated Hospital of Xuzhou Medical College, West Huaihai Road 99 (221002), Xuzhou, Jiangsu, PR China. E-mail: [email protected]
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.06.038
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2670
Transplantation Proceedings, 40, 2670 –2673 (2008)
ENDOTHELIAL INJURY
2671
Fig 1. Effect of methotrexate and cyclophosphamide on endothelial cells and T lymphocytes. A. Methotrexate group. B. Cyclophosphamide group.
damaged factor during HSCT is unknown. In the present study, we examined the role of single damaging factor— conventional chemotherapeutic agents used in HSCT (methotrexate or cyclophosphamide)—in relation to the time point conditioning for a HSCT with respect to endothelial injury and its functional consequences on GVHD. MATERIALS AND METHODS Mice and Reagents Six-week-old female Balb/c mice were purchased from the Animal Center in Xuzhou medical college (Jiangsu). These studies approved by our Institutional Animal Care and Use Committee housed and fed hosts in accordance with our animal center guidelines. PE-Cy5– conjugated rat anti-mouse CD45 monoclonal antibody and flourescein isothiocyanate (FITC)-conjugated rat anti-mouse CD31 monoclonal antibody were purchased from BD Pharmingen. A PE-conjugated rat anti-mouse CD133 monoclonal antibody was purchased from eBioscience.
Treatment With Methotrexate and Cyclophosphamide Three groups of mice were studied as for endothelial injury during bone marrow transplantation: methotrexate group (15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, and 11); cyclophosphamide group (60 mg/kg on days 1 and 2), and phosphate-buffered saline (control). The drugs were administered by intraperitoneal injection.
Monitoring the Circulating Endothelial Cells and CD4⫹/CD8⫹ T Cells Blood samples were obtained from mice on days 1, 3, 5, 7, 9, 11, 16, 21, and 26. Each sample was divided. One was incubated for 20 minutes at 4°C with PE-Cy5– conjugated rat anti-mouse CD45 monoclonal antibody, FITC-conjugated rat anti-mouse CD31 monoclonal antibody, or PE-conjugated rat anti-mouse CD133 monoclonal antibody. The other was incubated for 20 minutes at 4°C with FITC-conjugated rat anti-mouse CD3 and PE-conjugated rat anti-mouse CD4 monoclonal antibody/PE-conjugated rat antimouse CD8 monoclonal antibody monoclonal antibody. The ECs (CD133⫺, CD31⫹, CD45low)7 and CD3⫹CD4⫹/CD3⫹CD8⫹ T lymphocytes were enumerated by fluorescent activated cell sorting.
Tissue Harvest and Fixation Liver, skin, and intestinal tissues were harvested from mice on days 1, 3, 5, 7, and 9. A systemic blood vessel was flushed first with physiologic solution to clear the blood from the tissue, then perfused with fixation solution containing 2.5% glutaraldehyde and
1% paraform. The tissue specimen for ultrastructural or endothelial integrity by transmission electron microscopy was fixed with 2.5% glutaraldehyde, and that for morphology of tissue was fixed in 4% paraformaldehyde.
Assessing Morphologic Changes To assess morphologic changes, 4-m paraffin sections were stained with hematoxylin and eosin for histologic examination. The changes in endothelium and the necrotic tissue were assessed by light microscopy.
Statistical Analysis Data were expressed as mean values ⫾ standard deviations with P ⬍ .05 considered significant.
RESULTS Pristine Elevation of Circulating Endothelial Cells After Conditioning
The numbers of circulating ECs in the methotrexate group or the cyclophosphamide group were elevated abruptly at day 3 and day 1, respectively, gradually decreasing thereafter. Before they decreased, the number of cells in the methotrexate group were high for 13 days (Fig 1). Meanwhile, in both groups there was also a decrease in cells, especially CD8⫹ T cell. The ratio of CD4⫹ and CD8⫹ T cells increased gradually up to a peak, and thereafter recovered to normal levels. The time courses in both groups were different. In the methotrexate group, the ratio of T-lymphocyte subpopulations increased at day 7 and peaked at day 11. In the cyclophosphamide group, the time course was earlier— 4 days (Fig 1). We concluded that the immunosuppressive effects for these drugs occurred later than the endothelium injury. Changes in Morphology
Under light microscopy, the histologic changes occurred 6 days after conditioning. It was obviously different between the two groups. In the cyclophosphamide group, there was significant apomorphosis, necrosis, and thrombosis. In contrast, there were no visible histopathologic changes of ECs, except edema of parenchymal cells in the methotrexate group (Fig 2). DISCUSSION
Conditioning and immunosuppression are the key process in HSCT. However, dysfunction and failure are always the
2672
ZENG, YAN, DING ET AL
Fig 2. Pathologic changes of liver and cutaneous post conditioning. A. Liver from the methotrexate group. Hepatocytes were extensively hydropic, although the endothelium showed no visible pathologic changes (original magnification ⫻400). B. Liver from the cyclophosphamide group. Severe hydrops, thrombogenesis, and necrosis are present. C. Skin from the Methotrexate group (original magnification ⫻1000) Interstitium and endothelium showed no visible pathologic changes. D. Skin from the cyclophosphamide group (original magnification ⫻400). Severe hemorrhage is present.
challenges during transplantation. Previous documents have shown that excessive chemotherapy did not contribute to graft survival, but that high-dose immunosuppressants are responsible for infection. Methotrexate and cyclophosphamide, two conventional chemotherapeutic agents used in HSCT, depress or relieve rejection and GVHD. Our data showed that the two drug quickly lead to T-lymphocyte depletion, especially CD8⫹ T lymphocytes, which were predominant after conditioning for 1 week. A remarkable discovery was circulating ECs elevated before the change in T-lymphocyte subpopulations. Previous studies8 have reported that conditioning may damage the endothelium using circulating ECs as a marker of endothelial damage in allogeneic HSCT. Using this marker, we monitored both cyclophosphamide- and methotrexate-damaged endothelium before immune cells. This mechanism needs further investigation. It is well known that an integrated endothelium is the precondition of organ function. The endothelial monolayer at the interface between the extravascular space and the blood plays a crucial role in vascular homeostasis.9 The endothelial monolayer helps to maintain the antithrombotic, anti-inflammatory state of the microvascular bed. Additionally, it controls the tone and proliferative state of the underlying vascular smooth muscle cells. The loss of the endothelial barrier function is associated with severe consequences: tissue edema formation, thrombus formation, neointimal thickening, and abnormal responses to endothelium-dependent agonists.10
To understand the origin of increasing ECs in peripheral blood, we detected a change in morphology. There was a more significant EC degeneration with an increasing diastem between ECs and basilemma. Thrombus formation suggested that circulating ECs, at least partly, came from the damaged endothelium. It further confirmed that the number of circulatory ECs may be considered to be an important indicator of endothelial damage. From these data, we also discovered that the elevation of circulating ECs preceded histologic damage, providing an early marker of slight damage, which might be reversed by timely intervention. The reversibility of the endothelial injury reflects its severity as well as the capacity of cells to undergo the repair necessary for reconstitution of the endothelial barrier. Recovery of endothelial integrity after vascular injury is vital for endothelial barrier function and vascular homeostasis. Hence, further investigations are needed to determine whether circulating ECs are a marker of preservation or repair. REFERENCES 1. Biedermann BC: Vascular endothelial cells: an interesting immunological barrier. Schweiz Med Wochenschr 129:1712, 1999 2. Shimoni A, Yeshurun M, Hardan I, et al: Thrombotic microangiopathy after allogeneic stem cell transplantation in the era of reduced-intensity conditioning: the incidence is not reduced. Biol Blood Marrow Transplant 10:484, 2004 3. Potenza MA, Marasciuolo FL, Chieppa DM, et al: Insulin resistance in spontaneously hypertensive rats is associated with endo-
ENDOTHELIAL INJURY thelial dysfunction characterized by imbalance between NO and ET-1 production. Am J Physiol Heart Circ Physiol 289:H813, 2005 4. Paris F, Fuks Z, Kang A, et al: Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. Science 293:293, 2001 5. Ertault-Daneshpouy M, Leboeuf C, Lemann M, et al: Pericapillary hemorrhage as criterion of severe human digestive graftversus-host disease. Blood 5:1548, 2003 6. Ertault-Daneshpouy M, Leboeuf C, Lemann M, et al: Pericapillary hemorrhage as criterion of severe human digestive graftversus-host disease Blood 103:4681, 2004 7. Duda DG, Cohen KS, di Tomaso E, et al: Differential CD146 expression on circulating versus tissue endothelial cells in rectal
2673 cancer patients: implications for circulating endothelial and progenitor cells as biomarkers for antiangiogenic therapy. J Clin Oncol 24:1449, 2006 8. Woywodt A, Scheer J, Hambach L, et al: Circulating endothelial cells as a marker of endothelial damage in allogeneic hematopoietic stem cell transplantation. Blood 103:3603, 2004 9. Cines DB, Pollak ES, Buck CA, et al: Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 91:3527, 1998 10. McFadden EP, Bauters C, Lablanche JM, et al: Response of human coronary arteries to serotonin after injury by coronary angioplasty. Circulation 88:2076, 1993