Evaluation of Angiogenesis and Vascular Endothelial Growth Factor Expression in the Bone Marrow of Patients with Aplastic Anemia
2006; Elsevier BV; Volume: 168; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.050034
ISSN1525-2191
AutoresWolfgang Füreder, Maria‐Theresa Krauth, Wolfgang R. Sperr, Karoline Sonneck, Ingrid Simonitsch‐Klupp, Leonhard Müllauer, Michael Willmann, Hans‐Peter Horny, Peter Valent,
Tópico(s)Mesenchymal stem cell research
ResumoIt is generally appreciated that bone marrow function and growth of myelopoietic cells depends on an intact microvasculature. A pivotal regulator of angiogenesis is vascular endothelial growth factor (VEGF). Here, we describe analysis of VEGF expression and microvessel density in the bone marrow of patients with aplastic anemia by immunohistochemistry. Bone marrow was examined at diagnosis and at the time of hematological remission after immunosuppressive therapy using anti-thymocyte globulin, cyclosporin A, and glucocorticoids or allogeneic stem cell transplantation. At diagnosis, both VEGF expression and microvessel density were found to be significantly lower in aplastic anemia compared to normal bone marrow (aplastic anemia, 1.1 ± 0.7 events per field, versus controls, 5.9 ± 3.0 events per field; P < 0.05). In response to successful therapy, VEGF and microvessel density in the bone marrow increased substantially. Serum VEGF levels were also found to be significantly lower at diagnosis in aplastic anemia compared to healthy controls (aplastic anemia, 51 ± 35 pg/ml versus controls, 444 ± 220 pg/ml; P < 0.05). VEGF in the serum increased substantially after successful immunosuppressive therapy or stem cell transplantation (P < 0.05). Taken together, these data show that aplastic anemia is associated with reduced angiogenesis and reduced VEGF expression. It is generally appreciated that bone marrow function and growth of myelopoietic cells depends on an intact microvasculature. A pivotal regulator of angiogenesis is vascular endothelial growth factor (VEGF). Here, we describe analysis of VEGF expression and microvessel density in the bone marrow of patients with aplastic anemia by immunohistochemistry. Bone marrow was examined at diagnosis and at the time of hematological remission after immunosuppressive therapy using anti-thymocyte globulin, cyclosporin A, and glucocorticoids or allogeneic stem cell transplantation. At diagnosis, both VEGF expression and microvessel density were found to be significantly lower in aplastic anemia compared to normal bone marrow (aplastic anemia, 1.1 ± 0.7 events per field, versus controls, 5.9 ± 3.0 events per field; P < 0.05). In response to successful therapy, VEGF and microvessel density in the bone marrow increased substantially. Serum VEGF levels were also found to be significantly lower at diagnosis in aplastic anemia compared to healthy controls (aplastic anemia, 51 ± 35 pg/ml versus controls, 444 ± 220 pg/ml; P < 0.05). VEGF in the serum increased substantially after successful immunosuppressive therapy or stem cell transplantation (P < 0.05). Taken together, these data show that aplastic anemia is associated with reduced angiogenesis and reduced VEGF expression. Aplastic anemia (AA) is a life-threatening disorder characterized by a deficiency of pluripotent hematopoietic progenitor cells with consecutive bone marrow (BM) aplasia and peripheral pancytopenia.1Ehrlich P Über einen Fall von Anämie mit Bemerkungen über regenerative Veränderungen des Knochenmarks.Charité-Ann. 1888; 13: 301-309Google Scholar, 2Young NS Aplastic anemia.Lancet. 1995; 346: 2282-2332Crossref Scopus (66) Google Scholar, 3Young NS Maciejewski J The pathophysiology of acquired aplastic anemia.N Engl J Med. 1997; 336: 1365-1372Crossref PubMed Scopus (4) Google Scholar The histological hallmark of the disease is the empty marrow that contains normal amounts of fat cells but is more or less depleted of myelopoietic progenitor cells, erythroid cells, and megakaryocytes.3Young NS Maciejewski J The pathophysiology of acquired aplastic anemia.N Engl J Med. 1997; 336: 1365-1372Crossref PubMed Scopus (4) Google Scholar, 4Maciejewski JP Selleri C Sato T Anderson S Young NS A severe and consistent deficit in marrow and circulating primitive hematopoietic cells (long-term culture-initiating cells) in acquired aplastic anemia.Blood. 1996; 88: 1983-1991PubMed Google Scholar, 5Schrezenmeier H Jenal M Herrmann F Heimpel H Raghavachar A Quantitative analysis of cobblestone area-forming cells in bone marrow of patients with aplastic anemia by limiting dilution assay.Blood. 1996; 88: 4474-4480PubMed Google Scholar In a subset of patients, pancytopenia and depletion of myeloid cells in the BM are severe, denoting severe aplastic anemia (sAA).3Young NS Maciejewski J The pathophysiology of acquired aplastic anemia.N Engl J Med. 1997; 336: 1365-1372Crossref PubMed Scopus (4) Google Scholar, 4Maciejewski JP Selleri C Sato T Anderson S Young NS A severe and consistent deficit in marrow and circulating primitive hematopoietic cells (long-term culture-initiating cells) in acquired aplastic anemia.Blood. 1996; 88: 1983-1991PubMed Google Scholar, 5Schrezenmeier H Jenal M Herrmann F Heimpel H Raghavachar A Quantitative analysis of cobblestone area-forming cells in bone marrow of patients with aplastic anemia by limiting dilution assay.Blood. 1996; 88: 4474-4480PubMed Google Scholar When untreated, patients with AA have a grave prognosis.6Williams DM Lynch RE Cartwright GE Prognostic factors in aplastic anaemia.Clin Haematol. 1978; 7: 467-474PubMed Google Scholar, 7Lynch RE Williams DM Reading JC Cartwright GE The prognosis in aplastic anemia.Blood. 1975; 45: 517-528PubMed Google Scholar Current treatment options include hematopoietic stem cell transplantation or therapy with anti-thymocyte globulin (ATG) combined with cyclosporine A (CSA) and glucocorticoids.8Thomas ED Storb R Fefer A Slichter SJ Bryant JI Buckner CD Neiman PE Clift RA Funk DD Lerner KE Aplastic anaemia treated by marrow transplantation.Lancet. 1972; 1: 284-289Abstract PubMed Scopus (183) Google Scholar, 9Frickhofen N Kaltwasser JP Schrezenmeier H Raghavachar A Vogt HG Herrmann F Freund M Meusers P Salama A Heimpel H Treatment of aplastic anemia with antilymphocyte globulin and methylprednisolone with or without cyclosporine. The German Aplastic Anemia Study Group.N Engl J Med. 1991; 324: 1297-1304Crossref PubMed Scopus (376) Google Scholar, 10Gluckman E Rokicka-Milewska R Hann I Nikiforakis E Tavakoli F Cohen-Scali S Bacigalupo A European Group for Blood and Marrow Transplantation Working Party for Severe Aplastic Anemia: results and follow-up of a phase III randomized study of recombinant human-granulocyte stimulating factor as support for immunosuppressive therapy in patients with severe aplastic anaemia.Br J Haematol. 2002; 119: 1075-1082Crossref PubMed Scopus (90) Google Scholar, 11Frickhofen N Heimpel H Kaltwasser JP Schrezenmeier H German Aplastic Anemia Study Group: antithymocyte globulin with or without cyclosporin A: 11-year follow-up of a randomized trial comparing treatments of aplastic anemia.Blood. 2003; 101: 1236-1242Crossref PubMed Scopus (277) Google Scholar In response to these therapies, the majority of all patients with AA can be cured.8Thomas ED Storb R Fefer A Slichter SJ Bryant JI Buckner CD Neiman PE Clift RA Funk DD Lerner KE Aplastic anaemia treated by marrow transplantation.Lancet. 1972; 1: 284-289Abstract PubMed Scopus (183) Google Scholar, 9Frickhofen N Kaltwasser JP Schrezenmeier H Raghavachar A Vogt HG Herrmann F Freund M Meusers P Salama A Heimpel H Treatment of aplastic anemia with antilymphocyte globulin and methylprednisolone with or without cyclosporine. The German Aplastic Anemia Study Group.N Engl J Med. 1991; 324: 1297-1304Crossref PubMed Scopus (376) Google Scholar, 10Gluckman E Rokicka-Milewska R Hann I Nikiforakis E Tavakoli F Cohen-Scali S Bacigalupo A European Group for Blood and Marrow Transplantation Working Party for Severe Aplastic Anemia: results and follow-up of a phase III randomized study of recombinant human-granulocyte stimulating factor as support for immunosuppressive therapy in patients with severe aplastic anaemia.Br J Haematol. 2002; 119: 1075-1082Crossref PubMed Scopus (90) Google Scholar, 11Frickhofen N Heimpel H Kaltwasser JP Schrezenmeier H German Aplastic Anemia Study Group: antithymocyte globulin with or without cyclosporin A: 11-year follow-up of a randomized trial comparing treatments of aplastic anemia.Blood. 2003; 101: 1236-1242Crossref PubMed Scopus (277) Google Scholar So far, little is known about the pathogenesis of AA. Based on laboratory data and beneficial effects of immunosuppressive therapies, an abnormal function of the immune system has been discussed as contributing to the pathogenesis of AA.2Young NS Aplastic anemia.Lancet. 1995; 346: 2282-2332Crossref Scopus (66) Google Scholar, 3Young NS Maciejewski J The pathophysiology of acquired aplastic anemia.N Engl J Med. 1997; 336: 1365-1372Crossref PubMed Scopus (4) Google Scholar, 8Thomas ED Storb R Fefer A Slichter SJ Bryant JI Buckner CD Neiman PE Clift RA Funk DD Lerner KE Aplastic anaemia treated by marrow transplantation.Lancet. 1972; 1: 284-289Abstract PubMed Scopus (183) Google Scholar, 9Frickhofen N Kaltwasser JP Schrezenmeier H Raghavachar A Vogt HG Herrmann F Freund M Meusers P Salama A Heimpel H Treatment of aplastic anemia with antilymphocyte globulin and methylprednisolone with or without cyclosporine. The German Aplastic Anemia Study Group.N Engl J Med. 1991; 324: 1297-1304Crossref PubMed Scopus (376) Google Scholar, 10Gluckman E Rokicka-Milewska R Hann I Nikiforakis E Tavakoli F Cohen-Scali S Bacigalupo A European Group for Blood and Marrow Transplantation Working Party for Severe Aplastic Anemia: results and follow-up of a phase III randomized study of recombinant human-granulocyte stimulating factor as support for immunosuppressive therapy in patients with severe aplastic anaemia.Br J Haematol. 2002; 119: 1075-1082Crossref PubMed Scopus (90) Google Scholar, 11Frickhofen N Heimpel H Kaltwasser JP Schrezenmeier H German Aplastic Anemia Study Group: antithymocyte globulin with or without cyclosporin A: 11-year follow-up of a randomized trial comparing treatments of aplastic anemia.Blood. 2003; 101: 1236-1242Crossref PubMed Scopus (277) Google Scholar, 12Hinterberger W Adolf G Aichinger G Dudczak R Geissler K Hocker P Huber C Kalhs P Knapp W Koller U Further evidence for lymphokine overproduction in severe aplastic anemia.Blood. 1988; 72: 266-272PubMed Google Scholar, 13Nakao S Takami A Takamatsu H Zeng W Sugimori N Yamazaki H Miura Y Ueda M Shiobara S Yoshioka T Kaneshige T Yasukawa M Matsuda T Isolation of a T-cell clone showing HLA-DRB1*0405-restricted cytotoxicity for hematopoietic cells in a patient with aplastic anemia.Blood. 1997; 89: 3691-3699PubMed Google Scholar Thus, previous data have supported the notion that cytotoxic T cells may play a role in the development of AA.2Young NS Aplastic anemia.Lancet. 1995; 346: 2282-2332Crossref Scopus (66) Google Scholar, 3Young NS Maciejewski J The pathophysiology of acquired aplastic anemia.N Engl J Med. 1997; 336: 1365-1372Crossref PubMed Scopus (4) Google Scholar, 12Hinterberger W Adolf G Aichinger G Dudczak R Geissler K Hocker P Huber C Kalhs P Knapp W Koller U Further evidence for lymphokine overproduction in severe aplastic anemia.Blood. 1988; 72: 266-272PubMed Google Scholar, 13Nakao S Takami A Takamatsu H Zeng W Sugimori N Yamazaki H Miura Y Ueda M Shiobara S Yoshioka T Kaneshige T Yasukawa M Matsuda T Isolation of a T-cell clone showing HLA-DRB1*0405-restricted cytotoxicity for hematopoietic cells in a patient with aplastic anemia.Blood. 1997; 89: 3691-3699PubMed Google Scholar More recently, it has been suggested that humoral components of the immune system, including antibodies against cellular proteins, are also involved in the pathogenesis of AA.14Hirano N Butler MO Von Bergwelt-Baildon MS Maecker B Schultze JL O'Connor KC Schur PH Kojima S Guinan EC Nadler LM Autoantibodies frequently detected in patients with aplastic anemia.Blood. 2003; 102: 4567-4575Crossref PubMed Scopus (97) Google Scholar Another pathogenetic concept proposed in the context of AA, is related to the BM microenvironment, which consists of endosteal cells, macrophages, fat cells, fibroblasts, and microvascular endothelial cells.15Holmberg LA Seidel K Leisenring W Torok-Storb B Aplastic anemia: analysis of stromal cell function in long-term marrow cultures.Blood. 1994; 84: 3685-3690PubMed Google Scholar, 16Juneja HS Gardner FH Functionally abnormal marrow stromal cells in aplastic anemia.Exp Hematol. 1985; 13: 194-199PubMed Google Scholar In fact, it has been suggested that an abnormal (decreased) growth and function of such microenvironmental cells in the BM may contribute to the development of BM aplasia in patients with AA.15Holmberg LA Seidel K Leisenring W Torok-Storb B Aplastic anemia: analysis of stromal cell function in long-term marrow cultures.Blood. 1994; 84: 3685-3690PubMed Google Scholar, 16Juneja HS Gardner FH Functionally abnormal marrow stromal cells in aplastic anemia.Exp Hematol. 1985; 13: 194-199PubMed Google Scholar The microvasculature is an essential component of the microenvironment in diverse organs and has been described as a critical target in various disorders. Likewise, the BM microvasculature is considered to play an important role in hematopoietic neoplasms.17Pruneri G Bertolini F Soligo D Carboni N Cortelezzi A Ferrucci PF Buffa R Lambertenghi-Deliliers G Pezzella F Angiogenesis in myelodysplastic syndromes.Br J Cancer. 1999; 81: 1398-1401Crossref PubMed Scopus (243) Google Scholar, 18Padro T Ruiz S Bieker R Burger H Steins M Kienast J Buchner T Berdel WE Mesters RM Increased angiogenesis in the bone marrow of patients with acute myeloid leukemia.Blood. 2000; 95: 2637-2644PubMed Google Scholar, 19Aguayo A Kantarjian H Manshouri T Gidel C Estey E Thomas D Koller C Estrov Z O'Brian S Keating M Freireich E Albitar M Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes.Blood. 2000; 96: 2240-2245PubMed Google Scholar, 20Mesa R Hanson C Rajkumar S Schroeder G Tefferi A Evaluation and clinical correlations of bone marrow angiogenesis in myelofibrosis with myeloid metaplasia.Blood. 2000; 96: 3374-3380PubMed Google Scholar, 21Wimazal F Jordan JH Sperr WR Chott A Dabbass S Lechner K Horny HP Valent P Increased angiogenesis in the bone marrow of patients with systemic mastocytosis.Am J Pathol. 2002; 160: 1639-1645Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 22Perez-Atayde AR Sallan SE Tedrow U Connors S Allred E Folkman J Spectrum of tumor angiogenesis in the bone marrow of children with acute lymphoblastic leukemia.Am J Pathol. 1997; 150: 815-821PubMed Google Scholar, 23Ghannadan M Wimazal F Simonitsch I Sperr WR Mayerhofer M Sillaber C Hauswirth AW Gadner H Chott A Horny HP Lechner K Valent P Immunohistochemical detection of VEGF in the bone marrow of patients with acute myeloid leukemia (AML): correlation between VEGF expression and the FAB category.Am J Clin Pathol. 2003; 119: 663-671Crossref PubMed Scopus (36) Google Scholar, 24Krauth M Simonitsch I Aichberger K Mayerhofer M Sperr WR Sillaber C Schneeweiss B Mann G Gadner H Valent P Immunohistochemical detection of VEGF in the bone marrow of patients with chronic myeloid leukemia and correlation with the phase of disease.Am J Clin Pathol. 2004; 121: 473-481Crossref PubMed Scopus (30) Google Scholar Thus, in most BM neoplasms including myeloid leukemias and myelodysplastic syndromes, an enhanced BM angiogenesis has been described.17Pruneri G Bertolini F Soligo D Carboni N Cortelezzi A Ferrucci PF Buffa R Lambertenghi-Deliliers G Pezzella F Angiogenesis in myelodysplastic syndromes.Br J Cancer. 1999; 81: 1398-1401Crossref PubMed Scopus (243) Google Scholar, 18Padro T Ruiz S Bieker R Burger H Steins M Kienast J Buchner T Berdel WE Mesters RM Increased angiogenesis in the bone marrow of patients with acute myeloid leukemia.Blood. 2000; 95: 2637-2644PubMed Google Scholar, 19Aguayo A Kantarjian H Manshouri T Gidel C Estey E Thomas D Koller C Estrov Z O'Brian S Keating M Freireich E Albitar M Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes.Blood. 2000; 96: 2240-2245PubMed Google Scholar, 20Mesa R Hanson C Rajkumar S Schroeder G Tefferi A Evaluation and clinical correlations of bone marrow angiogenesis in myelofibrosis with myeloid metaplasia.Blood. 2000; 96: 3374-3380PubMed Google Scholar, 23Ghannadan M Wimazal F Simonitsch I Sperr WR Mayerhofer M Sillaber C Hauswirth AW Gadner H Chott A Horny HP Lechner K Valent P Immunohistochemical detection of VEGF in the bone marrow of patients with acute myeloid leukemia (AML): correlation between VEGF expression and the FAB category.Am J Clin Pathol. 2003; 119: 663-671Crossref PubMed Scopus (36) Google Scholar, 24Krauth M Simonitsch I Aichberger K Mayerhofer M Sperr WR Sillaber C Schneeweiss B Mann G Gadner H Valent P Immunohistochemical detection of VEGF in the bone marrow of patients with chronic myeloid leukemia and correlation with the phase of disease.Am J Clin Pathol. 2004; 121: 473-481Crossref PubMed Scopus (30) Google Scholar To a certain degree, the same may hold true for reactive disease states (systemic inflammation, chronic infection) in which enhanced BM function, ie, growth of progenitor cells in BM cavities, is required for the extra production of granulomonocytic effector cells. A central regulator of BM angiogenesis is vascular endothelial growth factor (VEGF).17Pruneri G Bertolini F Soligo D Carboni N Cortelezzi A Ferrucci PF Buffa R Lambertenghi-Deliliers G Pezzella F Angiogenesis in myelodysplastic syndromes.Br J Cancer. 1999; 81: 1398-1401Crossref PubMed Scopus (243) Google Scholar, 18Padro T Ruiz S Bieker R Burger H Steins M Kienast J Buchner T Berdel WE Mesters RM Increased angiogenesis in the bone marrow of patients with acute myeloid leukemia.Blood. 2000; 95: 2637-2644PubMed Google Scholar, 19Aguayo A Kantarjian H Manshouri T Gidel C Estey E Thomas D Koller C Estrov Z O'Brian S Keating M Freireich E Albitar M Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes.Blood. 2000; 96: 2240-2245PubMed Google Scholar, 20Mesa R Hanson C Rajkumar S Schroeder G Tefferi A Evaluation and clinical correlations of bone marrow angiogenesis in myelofibrosis with myeloid metaplasia.Blood. 2000; 96: 3374-3380PubMed Google Scholar, 21Wimazal F Jordan JH Sperr WR Chott A Dabbass S Lechner K Horny HP Valent P Increased angiogenesis in the bone marrow of patients with systemic mastocytosis.Am J Pathol. 2002; 160: 1639-1645Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 22Perez-Atayde AR Sallan SE Tedrow U Connors S Allred E Folkman J Spectrum of tumor angiogenesis in the bone marrow of children with acute lymphoblastic leukemia.Am J Pathol. 1997; 150: 815-821PubMed Google Scholar Among various VEGF species, VEGF-A appears to be expressed most abundantly in normal and neoplastic myeloid cells.17Pruneri G Bertolini F Soligo D Carboni N Cortelezzi A Ferrucci PF Buffa R Lambertenghi-Deliliers G Pezzella F Angiogenesis in myelodysplastic syndromes.Br J Cancer. 1999; 81: 1398-1401Crossref PubMed Scopus (243) Google Scholar, 18Padro T Ruiz S Bieker R Burger H Steins M Kienast J Buchner T Berdel WE Mesters RM Increased angiogenesis in the bone marrow of patients with acute myeloid leukemia.Blood. 2000; 95: 2637-2644PubMed Google Scholar, 19Aguayo A Kantarjian H Manshouri T Gidel C Estey E Thomas D Koller C Estrov Z O'Brian S Keating M Freireich E Albitar M Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes.Blood. 2000; 96: 2240-2245PubMed Google Scholar, 20Mesa R Hanson C Rajkumar S Schroeder G Tefferi A Evaluation and clinical correlations of bone marrow angiogenesis in myelofibrosis with myeloid metaplasia.Blood. 2000; 96: 3374-3380PubMed Google Scholar, 21Wimazal F Jordan JH Sperr WR Chott A Dabbass S Lechner K Horny HP Valent P Increased angiogenesis in the bone marrow of patients with systemic mastocytosis.Am J Pathol. 2002; 160: 1639-1645Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 22Perez-Atayde AR Sallan SE Tedrow U Connors S Allred E Folkman J Spectrum of tumor angiogenesis in the bone marrow of children with acute lymphoblastic leukemia.Am J Pathol. 1997; 150: 815-821PubMed Google Scholar Thus, in most instances, the levels of VEGF-A correlate with the extent of angiogenesis, ie, the BM microvessel density (MVD).20Mesa R Hanson C Rajkumar S Schroeder G Tefferi A Evaluation and clinical correlations of bone marrow angiogenesis in myelofibrosis with myeloid metaplasia.Blood. 2000; 96: 3374-3380PubMed Google Scholar, 21Wimazal F Jordan JH Sperr WR Chott A Dabbass S Lechner K Horny HP Valent P Increased angiogenesis in the bone marrow of patients with systemic mastocytosis.Am J Pathol. 2002; 160: 1639-1645Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 22Perez-Atayde AR Sallan SE Tedrow U Connors S Allred E Folkman J Spectrum of tumor angiogenesis in the bone marrow of children with acute lymphoblastic leukemia.Am J Pathol. 1997; 150: 815-821PubMed Google Scholar Within the hematopoietic system, VEGF is considered to be primarily produced in megakaryocytes and immature myeloid cells.24Krauth M Simonitsch I Aichberger K Mayerhofer M Sperr WR Sillaber C Schneeweiss B Mann G Gadner H Valent P Immunohistochemical detection of VEGF in the bone marrow of patients with chronic myeloid leukemia and correlation with the phase of disease.Am J Clin Pathol. 2004; 121: 473-481Crossref PubMed Scopus (30) Google Scholar, 25Mohle R Green D Moore MA Nachman RL Rafii S Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets.Proc Natl Acad Sci USA. 1997; 94: 663-668Crossref PubMed Scopus (624) Google Scholar Based on this hypothesis, it was of interest to learn whether BM angiogenesis and VEGF expression change in patients with AA. The specific aims of the present study were to examine BM angiogenesis and VEGF expression in patients with AA and to compare these values with those obtained in normal BM. In addition, we examined the effects of immunosuppressive therapy (with ATG, CSA, and glucocorticoids) or allogeneic stem cell transplantation on angiogenesis and VEGF expression. Eighteen patients with AA were examined in this study. BM specimens were obtained from the posterior iliac crest in all cases after informed consent was given. Additional diagnostic investigations included physical examination, ultrasound (spleen), complete blood count, and blood chemistry. The patients' characteristics are shown in Table 1. The diagnosis of AA was established according to published criteria.26Rozman C Marin P Nomdedeu B Montserrat E Criteria for severe aplastic anaemia.Lancet. 1987; 2: 955-957Abstract PubMed Scopus (20) Google Scholar, 27Camitta BM What is the definition of cure for aplastic anemia?.Acta Haematol. 2000; 103: 16-18Crossref PubMed Scopus (97) Google Scholar Diagnosis of sAA required a hypocellular BM (cellularity <30% according to Tuzuner and Bennett28Tuzuner N Bennett JM Reference standards for bone marrow cellularity.Leuk Res. 1994; 18: 645-647Abstract Full Text PDF PubMed Scopus (38) Google Scholar) and two or three of the following abnormal peripheral blood values: absolute neutrophil count <0.5 × 109/L, platelets <20 × 109/L, or anemia with reticulocytopenia of <20 × 109/L. The diagnosis of very severe aplastic anemia (vsAA) was established when sAA criteria were met and absolute neutrophil counts were <0.2 × 109/L. Nonsevere AA (nsAA) was defined as pancytopenia with hypocellular BM and peripheral blood counts not fulfilling criteria of sAA. Based on these criteria, 9 of 18 patients (50%) were classified as nsAA, 6 patients (33%) as sAA, and 3 patients (17%) as vsAA. In all but six patients, the BM was re-examined after immunosuppressive therapy (ATG, CSA, and glucocorticoids) or allogeneic stem cell transplantation.Table 1Patients' Characteristics and Results of Routine Laboratory InvestigationsNo.DiagnosisSexAge at diagnosis (years)WBC (×109/L)ANC (×109/L)Hb (g/dl)Reti (×109/L)Plt (×109/L)01nsAAF482.01.08.0314202nsAAF582.71.09.549703nsAAM343.62.36.2364204nsAAF373.62.09.6915905nsAAM252.10.67.0291106nsAAF661.1na8.4322807nsAAF373.00.89.1821608nsAAM312.50.712.7535209nsAAF463.41.78.8222410sAAM653.31.710.5181711sAAF342.11.88.413312sAAM201.70.28.0143313sAAM241.40.46.9351114sAAM580.80.36.741715sAAM282.00.28.6174416vsAAF422.30.19.6106617vsAAF651.00.011.4101718vsAAF792.00.19.21724nsAA, nonsevere aplastic anemia; sAA, severe aplastic anemia; vsAA, very severe aplastic anemia; WBC, white blood cell count; ANC, absolute neutrophil count; Hb, hemoglobin; Reti, reticulocytes; Plt, platelets. na, not available. Open table in a new tab nsAA, nonsevere aplastic anemia; sAA, severe aplastic anemia; vsAA, very severe aplastic anemia; WBC, white blood cell count; ANC, absolute neutrophil count; Hb, hemoglobin; Reti, reticulocytes; Plt, platelets. na, not available. Control BM specimens were obtained from three patients with non-Hodgkin's lymphoma, one with suspected myeloproliferative disease, one with suspected multiple myeloma, one with iron deficiency, and one with a local infection without myeloid hyperplasia. None of the control patients had signs of systemic inflammation or lymphoma-associated B symptoms. All patients gave informed consent before BM puncture. Patients were treated with ATG, CSA, and glucocorticoids according to published protocols8Thomas ED Storb R Fefer A Slichter SJ Bryant JI Buckner CD Neiman PE Clift RA Funk DD Lerner KE Aplastic anaemia treated by marrow transplantation.Lancet. 1972; 1: 284-289Abstract PubMed Scopus (183) Google Scholar, 9Frickhofen N Kaltwasser JP Schrezenmeier H Raghavachar A Vogt HG Herrmann F Freund M Meusers P Salama A Heimpel H Treatment of aplastic anemia with antilymphocyte globulin and methylprednisolone with or without cyclosporine. The German Aplastic Anemia Study Group.N Engl J Med. 1991; 324: 1297-1304Crossref PubMed Scopus (376) Google Scholar after written informed consent was obtained. Fourteen of the eighteen patients received horse ATG (15 mg/kg per day i.v. for 5 to 8 days) and one rabbit ATG (10 mg/kg per day for 5 days) as well as CSA per os (to reach a target serum CSA concentration of 100 to 200 ng/ml) and glucocorticoids (to prevent serum sickness). Nine patients received granulocyte-colony stimulating factor (G-CSF) subcutaneously at a dose of 5 μg/kg per day until neutrophil re-covery. In three patients, hematopoietic stem cell transplantation was performed using stem cells from an HLA-matched sibling donor after written informed consent was given. In all patients, the BM was examined by routine histology and cytology at diagnosis. In 12 patients, BM was analyzed before and after successful therapy. Immunohistochemistry was performed on paraffin-embedded, formalin-fixed BM biopsy sections (15 patients) using the indirect immunoperoxidase staining technique as described.21Wimazal F Jordan JH Sperr WR Chott A Dabbass S Lechner K Horny HP Valent P Increased angiogenesis in the bone marrow of patients with systemic mastocytosis.Am J Pathol. 2002; 160: 1639-1645Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 23Ghannadan M Wimazal F Simonitsch I Sperr WR Mayerhofer M Sillaber C Hauswirth AW Gadner H Chott A Horny HP Lechner K Valent P Immunohistochemical detection of VEGF in the bone marrow of patients with acute myeloid leukemia (AML): correlation between VEGF expression and the FAB category.Am J Clin Pathol. 2003; 119: 663-671Crossref PubMed Scopus (36) Google Scholar, 24Krauth M Simonitsch I Aichberger K Mayerhofer M Sperr WR Sillaber C Schneeweiss B Mann G Gadner H Valent P Immunohistochemical detection of VEGF in the bone marrow of patients with chronic myeloid leukemia and correlation with the phase of disease.Am J Clin Pathol. 2004; 121: 473-481Crossref PubMed Scopus (30) Google Scholar Endogenous peroxidase was blocked by methanol/H2O2. The CD34 monoclonal antibody (mAb) QBEND 10 (diluted 1:100) was purchased from Immunotech (Marseilles, France), an antibody against FVIII-related antigen (FVIII-rAG) from Dakopatts (Glostrup, Denmark) (diluted 1:400), and a rabbit anti-VEGF antibody (diluted 1:50) as well as a VEGF-blocking peptide from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies were diluted in 0.05 mol/L Tris-buffered saline (pH 7.5) plus 1% bovine serum albumin and applied for 60 minutes. In case of VEGF staining, BM sections were pretreated by microwave oven. In selected cases, the anti-VEGF antibody was preincubated with a VEGF-blocking peptide before staining. After washing, slides were incubated with biotinylated horse anti-mouse or biotinylated goat anti-rabbit IgG for 30 minutes, washed, and exposed to avidin-biotin-peroxidase or streptavidin-biotin-peroxidase complex for 30 minutes. 3-Amino-9-ethyl-carbazole was used as chromogen. Slides were counterstained in Mayer's hemalaun. Before quantification, VEGF-stained BM slides were carefully examined for internal positive controls (myeloid progenitors, megakaryocytes, plasma cells) and negative controls (erythrocytes, mature granulocytic cells). Only those slides that showed a homogenous staining pattern for VEGF and the appropriate result for internal (positive and negative) controls were subjected to quantitative analyses. Apart from hematopoietic cells, vessel-lining cells and stromal cells were also labeled by the anti-VEGF antibody, although most of the VEGF in the normal BM was found to be expressed by myeloid cells. Staining results obtained with the anti-VEGF antibody were quantified using an arbitrary score: 1, no reactivity found; 2, only a minority of nucleated cells reactive; 3, majority of all nucleated cells reacting with anti-VEGF; 4, strong expression of VEGF in most cells. The cellularity of the BM was quantified according to published guidelines.28Tuzuner N Bennett JM Reference standards for bone marrow cellularity.Leuk Res. 1994; 18: 645-647Abstract Full Text PDF PubMed Scopus (38) Google Scho
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