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Therapeutic angiogenesis by autologous transplantation of bone marrow mononuclear cells for peripheral artery disease
International Congress Series 1299 (2007) 203 – 209
www.ics-elsevier.com
Therapeutic angiogenesis by autologous transplantation of bone marrow mononuclear cells for peripheral artery disease Kazuhiro Nagai a,⁎, Ichiroh Matsumaru b , Takuya Fukushima c , Yasushi Miyazaki c , Hiroichiroh Yamaguchi b , Shimeru Kamihira a , Kiyoyuki Eishi b , Masao Tomonaga c a
b
Transfusion Service, Nagasaki University Hospital of Medicine and Dentistry, Nagasaki, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan Department of Cardiovascular Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan c Department of Molecular Medicine and Hematology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
Abstract. Medical management of the acute radiation injury might be an important area to which the procedures of regeneration medicine and stem cell therapy could contribute, since organ stem cells might be targets of radiation injury. Furthermore, since the recovery of blood flow is an important factor in the restoration of many tissues, therapeutic angiogenesis is assumed to become essential treatment in this field. In 2003 we started a project of therapeutic angiogenesis by implantation of autologous bone marrow mononuclear cells in conformity to the protocol of Therapeutic Angiogenesis Using Cell Transplantation trial, which treated patients with severe lower limb ischemia. Two cases of Buerger disease were treated and their symptoms were improved within 2–4 weeks after the transplantation. Angiography indicated that there emerged new blood vessels in patient's treated limb. Augmentation of blood flow was suggested by thermography in one case. In both cases, no serious adverse symptoms due to this treatment were observed. In the present trial, we could recognize effectiveness and safety of therapeutic angiogenesis. We need to improve the present procedure with introduction of evolving knowledge regarding mechanisms of physiological, patho-
⁎ Corresponding author. Tel.: +81 95 849 7455; fax: +81 95 849. E-mail address: [email protected] (K. Nagai). 0531-5131/ © 2007 Published by Elsevier B.V. doi:10.1016/j.ics.2006.09.003
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logical and utilizable therapeutic angiogenesis and should develop the best strategy of tissue regeneration medicine for victims of radiation exposure. © 2007 Published by Elsevier B.V. Keywords: Therapeutic angiogenesis; Bone marrow; Endothelial progenitor cell; Regeneration medicine; Acute radiation injury
1. Introduction In acute phase of radiation exposure, organ stem cells and rapidly dividing cells such as those of bone marrow (BM) or intestinal mucosa might be targets of radiation injury resulting in BM failure or gastrointestinal mucosal lesion [1]. Hematopoietic stem cell transplantation (HSCT) might rescue some patients with such hematological insufficiency. Thus, medical management of the acute radiation injury might be an important area to which procedures of regeneration medicine and stem cell therapy could contribute. Endothelial cell (EC) is also recognized to have sensitivity to ionizing radiation which causes changes of permeability, hypoxia, and apoptotic changes in exposed ECs [2]. These changes to ECs might cause microthrombs and vascular occlusion. As a result, these might cause vascular insufficiency in a number of tissues, and it is considered that damage of irradiated ECs might be an important pathological factor of emergence of skin ulcer or central nerves system syndrome caused by radiation exposure. Therefore, reconstruction of organ blood flow by therapeutic angiogenesis is assumed to become essential and common treatment in the medical care in victims of radiation exposure, especially in the restoration of many damaged tissues. Recently, endothelial progenitor cell (EPC) has been isolated from adult peripheral blood by Dr. Asahara's group [3]. EPCs are considered to share common stem/progenitor cells with HSCs and have been shown to derive mainly from BM and to incorporate into foci of physiological and pathological neovascularization. Presently, we intend to establish the strategy of regenerative medicine using stem cells for diverse medical areas, and especially, we are trying to apply this promising method for medical treatment of radiological casualties. In the present report, we demonstrated our experiences of therapeutic angiogenesis by autologous transplantation of BM mononuclear cell (BMMNC) and discussed its efficiency for treatment of radiation exposure victims. 2. Subjects and methods We have started a project of therapeutic angiogenesis by implantation of autologous BMMNCs under the attestation of the Ethical Committee of Nagasaki University Hospital since 2003. We are presently performing this project in conformity with the double-blind, randomized controlled protocol of TACT (Therapeutic Angiogenesis Using Cell Transplantation) trial [4], which was conducted by the Japanese cooperative group of Jichi Medical School, Kansai Medical School, and Kurume University School of Medicine, and which treated patients with severe lower limb ischemia (Fig. 1). Effectiveness of the treatment was evaluated by the following methods; improvement of symptoms, pain-free walking time using the treadmill test, thermography, testing of the
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Fig. 1. Procedure outline of therapeutic angiogenesis in accordance with TACT trial. With informed consent and after the evaluation in accordance with the eligibility criteria, BM cells were harvested from patient's iliac bone under general anesthesia. BMMNCs were then processed in closed system into the concentration of 3–7 × 107/mL by centrifugation cell-separator (COBE Spectra AUTO PBSC; GAMBRO), and this concentrated suspension of BMMNCs was implanted into approximately 50 points of patient's ischemic limb by intramuscular injection (injection volume: 1 ml/site).
ankle pressure index (API), transcutaneous oxygen pressure (TcO2), angiography, and magnetic resonance (MR) angiography. Simultaneously, we evaluated the quality of autologous marrow graft by in vitro quantification of colony forming unit-endothelial cell (CFU-EC) in patients' BMMNCs by EndoCult™ liquid culture system (StemCell Technologies, Vancouver, Canada) in accordance with manufacturer's instructions. Furthermore, we tested the frequency of EPCs in the grafts by immunophenotyping using specific monoclonal antibodies (MoAb) to CD34 and Flk-1 which were considered as markers of EPC. Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD34 (BD Medical Systems, Franklin Lakes, NJ, USA) and phycoerythrin (PE)-conjugated anti-Flk-1 (R&D Systems, Minneapolis, MN, USA), and analyzed by FACS Caliber™ (BD Medical Systems). 3. Results 3.1. Case 1 The first case was a 48-year-old male patient diagnosed of Buerger disease. He suffered from intermittent claudication, leg pain, and recurrent skin ulcer for 5 years. His disease was highly resistant to any given treatment like medication or bypass grafting surgery. Unfortunately, his left leg was amputated because of severe pain, deep skin ulcer with persisting infection at 5 years after onset. Since he also had leg pain and skin ulcer in his right leg simultaneously, we decided to perform therapeutic angiogenesis for his right leg. At this time, in his right leg, TcO2 was undetectable, and angiography and MR angiography indicated that markedly reduced blood flow due to obstruction of anterior
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tibial and dorsal pedal artery, and severe stenosis in posterior tibial artery in his right leg. As a result of the operation, in total, 6.89 × 109 BMMNCs were harvested and implanted into his right limb at 5.0 × 107 BMMNCs/mL/site. There was no remarkable complication due to the transplantation. Two weeks after the operation, improvement of leg pain was observed. Elevation of TcO2 level was observed just after 1 week of the transplantation and normalized at 4 weeks after, especially in dorsal region (Fig. 2A). At 4 and 6 weeks after the transplantation, MR angiography and angiography, respectively, revealed that there emerged new blood vessels of transplanted limb (Fig. 2B–E). One year after the transplantation, there was no recurrence of skin ulcer and he returned to social life. 3.2. Case 2 The second case was a 58-year-old male patient with diagnosis of Buerger disease. He suffered from intermittent claudication, leg pain and numbness in his left limb for 2 years.
Fig. 2. Improved findings in Case 1. (A) Findings of transcutaneous oxygen pressure (TcO2). (B–E) Findings of angiography at pre-transplantation (B and D) and those at 6 months after the treatment (C and E); (B and C) anterior and posterior tibial region; (D and E) pedal region. Emergence of fine new blood vessels in gastrocnemius and soleus muscle of transplanted limb, and improvement or recovery of blood vessel contrast in anterior and posterior tibial artery and pedal artery are observed after the transplantation.
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Repetitive bypass grafting surgery was performed, but gave him only insufficient result. Thus, we decided to perform therapeutic angiogenesis for his left limb. Angiography indicated obstruction of posterior tibial artery and dorsal pedal artery in his left limb. As a result of the operation, in total, 1.4 × 109 BMMNCs were harvested and implanted into his left limb at 60 points. There was no remarkable complication or adverse symptom due to the transplantation. Two weeks after the operation, numbness and leg pain were reduced. The findings of thermography revealed that augmentation of blood flow in patient's treated leg was suggested by elevation of skin temperature at 1 week after of the treatment. 3.3. EPC assay In the first and second cases, the frequencies of CFU-EC were 26.8 and 18.5/106 BMMNCs, respectively (Fig. 3A). These were almost equivalent results to those of healthy adult BM (19.0 ± 10.0/106 BMMNCs). Furthermore, FACS analysis revealed that frequency of EPCs in marrow graft of the first and second cases were 0.1% and 0.039%, respectively. These results were also equivalent to those of healthy adult BM (0.057± 0.016%). 3.4. Growth factors We investigated changes of serum level of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) which were both considered to play an important role
Fig. 3. Findings of EPC assay and cytokines. (A) A picture of representative colony forming unit-endothelial cell (CFU-EC) in Case 2. (B) Changes in serum level of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). VEGF and HGF were quantified by EIA and ELISA, respectively.
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in neovascularization. In both cases, serum level of VEGF was markedly elevated in posttransplantation phase (Fig. 3 B). 4. Discussion In the present study, we recognized the effectiveness and safety of therapeutic angiogenesis by implantation of autologous BMMNCs as indicated in the successful report from TACT trial [4]. Accumulation of clinical cases and large-scale clinical studies are necessary to clarify long-term verification of safety and effectiveness. Furthermore, we should be aware that there are some controversies in this procedure. The discovery of EPCs has drastically changed our understanding of adult blood vessel formation. The finding that EPCs home to sites of neovascularization and differentiate into ECs in situ is consistent with “vasuculogenesis,” a critical paradigm well described for embryonic neovascularization. On the other hand, the so-called “angiogenesis” are caused by activation of in situ ECs by various pro-angiogenic factors (i.e., VEGF, HGF, granulocyte-colony stimulating factor: G-CSF, platelet-derived growth factor-BB: PDGFBB, and matrix metalloproteinase 9: MMP-9, etc.). More recently, there are several studies that the therapeutic angiogenesis might function as an integrated therapy of in situ cells, cellular sources in transplanted BM, and pro-angiogenic factors, suggesting vascular restoration in treated limbs was achieved by collateral remodeling through paracrine systems [5]. Indeed, we could observe elevation of serum VEGF level in both cases in the post-transplant phase. Thus, in order to get the best of therapeutic efficacy, we should further investigate cellular and molecular mechanisms of the reconstruction of blood vessels, especially of arteriogenesis with maturation by acquisition of smooth muscle cell coat and extracellular matrix in physiological, pathological conditions, and in the process of utilizable therapeutic angiogenesis. Since there might be a danger of BM failure simultaneously in patients with radiation exposure, an availability of autologous marrow cells is limited in many exposured cases. Therefore, it is overwhelmingly likely that appropriately selected patients will undergo an allogeneic transplant procedure, although there are several barriers to allogeneic transplantation which limit its effectiveness, such as immunological effects between donor and recipient, and considerable time for donor search [6]. Furthermore, HSCT is presently recommended only for individuals who do not suffer significant injury to nonhematopoietic organs, since such multiple concomitant injuries to non-hematopoietic organs limit the effectiveness of HSCT [7]. On the other hand, in vivo evidence was recently reported which revealed neoendothelialization by donor-origin cells in a clinical case of the Tokai-mura nuclear accident which occurred in 1999 [8]. Therefore, our challenges in this field should include exploration of novel approaches which could expand the role of cell transplantation therapy in the victims of radiological events from reconstruction of hematopoietic function to restoration of non-hematopoietic organs. We should develop novel cellular sources and the best way of isolation and ex vivo expansion of stem cells and regulation of their survival and function following transplantation with the combined use of soluble factors and/or bioengineering techniques. Exploration of such new strategies might shed light on new directions for tissue regeneration medicine for victims of radiation exposure.
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References [1] K.L. Koenig, R.E. Goans, R.J. Hatchett, et al., Medical treatment of radiological casualties: current concepts, Ann. Emerg. Med. 45 (2005) 643–652. [2] L.F. Fajardo, M. Berthrong, Vascular lesions following radiation, Pathol. Annu. 23 (1988) 297–330. [3] T. Asahara, T. Murohara, A. Sullivan, et al., Isolation of putative progenitor endothelial cells for angiogenesis, Science 275 (1997) 964–967. [4] E. Tateishi-Yuyama, H. Matsubara, T. Murohara, et al., Therapeutic Angiogenesis Using Cell Transplantation (TACT) Study Investigators, Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomized controlled trial, Lancet 360 (2002) 427–435. [5] T. Kinnaird, E. Stabile, M.S. Burnett, et al., Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms, Circ. Res. 94 (2004) 678–685. [6] N. Dainiak, R.C. Ricks, The evolving role of haematopoietic cell transplantation in radiation injury: potentials and limitations, Br. J. Radiol., Suppl. 27 (2005) 169–174. [7] J.K. Waselenko, T.J. MacVittie, W.F. Blakely, et al., Strategic National Stockpile Radiation Working Group, Medical management of the acute radiation syndrome: recommendations of the Strategic National Stockpile Radiation Working Group, Ann. Intern. Med. 140 (2004) 1037–1051. [8] T. Suzuki, M. Nishida, S. Futami, et al., Neoendothelialization after peripheral blood stem cell transplantation in humans: a case report of a Tokaimura nuclear accident victim, Cardiovasc. Res. 58 (2003) 487–492.
www.ics-elsevier.com
Therapeutic angiogenesis by autologous transplantation of bone marrow mononuclear cells for peripheral artery disease Kazuhiro Nagai a,⁎, Ichiroh Matsumaru b , Takuya Fukushima c , Yasushi Miyazaki c , Hiroichiroh Yamaguchi b , Shimeru Kamihira a , Kiyoyuki Eishi b , Masao Tomonaga c a
b
Transfusion Service, Nagasaki University Hospital of Medicine and Dentistry, Nagasaki, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan Department of Cardiovascular Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan c Department of Molecular Medicine and Hematology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
Abstract. Medical management of the acute radiation injury might be an important area to which the procedures of regeneration medicine and stem cell therapy could contribute, since organ stem cells might be targets of radiation injury. Furthermore, since the recovery of blood flow is an important factor in the restoration of many tissues, therapeutic angiogenesis is assumed to become essential treatment in this field. In 2003 we started a project of therapeutic angiogenesis by implantation of autologous bone marrow mononuclear cells in conformity to the protocol of Therapeutic Angiogenesis Using Cell Transplantation trial, which treated patients with severe lower limb ischemia. Two cases of Buerger disease were treated and their symptoms were improved within 2–4 weeks after the transplantation. Angiography indicated that there emerged new blood vessels in patient's treated limb. Augmentation of blood flow was suggested by thermography in one case. In both cases, no serious adverse symptoms due to this treatment were observed. In the present trial, we could recognize effectiveness and safety of therapeutic angiogenesis. We need to improve the present procedure with introduction of evolving knowledge regarding mechanisms of physiological, patho-
⁎ Corresponding author. Tel.: +81 95 849 7455; fax: +81 95 849. E-mail address: [email protected] (K. Nagai). 0531-5131/ © 2007 Published by Elsevier B.V. doi:10.1016/j.ics.2006.09.003
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logical and utilizable therapeutic angiogenesis and should develop the best strategy of tissue regeneration medicine for victims of radiation exposure. © 2007 Published by Elsevier B.V. Keywords: Therapeutic angiogenesis; Bone marrow; Endothelial progenitor cell; Regeneration medicine; Acute radiation injury
1. Introduction In acute phase of radiation exposure, organ stem cells and rapidly dividing cells such as those of bone marrow (BM) or intestinal mucosa might be targets of radiation injury resulting in BM failure or gastrointestinal mucosal lesion [1]. Hematopoietic stem cell transplantation (HSCT) might rescue some patients with such hematological insufficiency. Thus, medical management of the acute radiation injury might be an important area to which procedures of regeneration medicine and stem cell therapy could contribute. Endothelial cell (EC) is also recognized to have sensitivity to ionizing radiation which causes changes of permeability, hypoxia, and apoptotic changes in exposed ECs [2]. These changes to ECs might cause microthrombs and vascular occlusion. As a result, these might cause vascular insufficiency in a number of tissues, and it is considered that damage of irradiated ECs might be an important pathological factor of emergence of skin ulcer or central nerves system syndrome caused by radiation exposure. Therefore, reconstruction of organ blood flow by therapeutic angiogenesis is assumed to become essential and common treatment in the medical care in victims of radiation exposure, especially in the restoration of many damaged tissues. Recently, endothelial progenitor cell (EPC) has been isolated from adult peripheral blood by Dr. Asahara's group [3]. EPCs are considered to share common stem/progenitor cells with HSCs and have been shown to derive mainly from BM and to incorporate into foci of physiological and pathological neovascularization. Presently, we intend to establish the strategy of regenerative medicine using stem cells for diverse medical areas, and especially, we are trying to apply this promising method for medical treatment of radiological casualties. In the present report, we demonstrated our experiences of therapeutic angiogenesis by autologous transplantation of BM mononuclear cell (BMMNC) and discussed its efficiency for treatment of radiation exposure victims. 2. Subjects and methods We have started a project of therapeutic angiogenesis by implantation of autologous BMMNCs under the attestation of the Ethical Committee of Nagasaki University Hospital since 2003. We are presently performing this project in conformity with the double-blind, randomized controlled protocol of TACT (Therapeutic Angiogenesis Using Cell Transplantation) trial [4], which was conducted by the Japanese cooperative group of Jichi Medical School, Kansai Medical School, and Kurume University School of Medicine, and which treated patients with severe lower limb ischemia (Fig. 1). Effectiveness of the treatment was evaluated by the following methods; improvement of symptoms, pain-free walking time using the treadmill test, thermography, testing of the
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Fig. 1. Procedure outline of therapeutic angiogenesis in accordance with TACT trial. With informed consent and after the evaluation in accordance with the eligibility criteria, BM cells were harvested from patient's iliac bone under general anesthesia. BMMNCs were then processed in closed system into the concentration of 3–7 × 107/mL by centrifugation cell-separator (COBE Spectra AUTO PBSC; GAMBRO), and this concentrated suspension of BMMNCs was implanted into approximately 50 points of patient's ischemic limb by intramuscular injection (injection volume: 1 ml/site).
ankle pressure index (API), transcutaneous oxygen pressure (TcO2), angiography, and magnetic resonance (MR) angiography. Simultaneously, we evaluated the quality of autologous marrow graft by in vitro quantification of colony forming unit-endothelial cell (CFU-EC) in patients' BMMNCs by EndoCult™ liquid culture system (StemCell Technologies, Vancouver, Canada) in accordance with manufacturer's instructions. Furthermore, we tested the frequency of EPCs in the grafts by immunophenotyping using specific monoclonal antibodies (MoAb) to CD34 and Flk-1 which were considered as markers of EPC. Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD34 (BD Medical Systems, Franklin Lakes, NJ, USA) and phycoerythrin (PE)-conjugated anti-Flk-1 (R&D Systems, Minneapolis, MN, USA), and analyzed by FACS Caliber™ (BD Medical Systems). 3. Results 3.1. Case 1 The first case was a 48-year-old male patient diagnosed of Buerger disease. He suffered from intermittent claudication, leg pain, and recurrent skin ulcer for 5 years. His disease was highly resistant to any given treatment like medication or bypass grafting surgery. Unfortunately, his left leg was amputated because of severe pain, deep skin ulcer with persisting infection at 5 years after onset. Since he also had leg pain and skin ulcer in his right leg simultaneously, we decided to perform therapeutic angiogenesis for his right leg. At this time, in his right leg, TcO2 was undetectable, and angiography and MR angiography indicated that markedly reduced blood flow due to obstruction of anterior
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tibial and dorsal pedal artery, and severe stenosis in posterior tibial artery in his right leg. As a result of the operation, in total, 6.89 × 109 BMMNCs were harvested and implanted into his right limb at 5.0 × 107 BMMNCs/mL/site. There was no remarkable complication due to the transplantation. Two weeks after the operation, improvement of leg pain was observed. Elevation of TcO2 level was observed just after 1 week of the transplantation and normalized at 4 weeks after, especially in dorsal region (Fig. 2A). At 4 and 6 weeks after the transplantation, MR angiography and angiography, respectively, revealed that there emerged new blood vessels of transplanted limb (Fig. 2B–E). One year after the transplantation, there was no recurrence of skin ulcer and he returned to social life. 3.2. Case 2 The second case was a 58-year-old male patient with diagnosis of Buerger disease. He suffered from intermittent claudication, leg pain and numbness in his left limb for 2 years.
Fig. 2. Improved findings in Case 1. (A) Findings of transcutaneous oxygen pressure (TcO2). (B–E) Findings of angiography at pre-transplantation (B and D) and those at 6 months after the treatment (C and E); (B and C) anterior and posterior tibial region; (D and E) pedal region. Emergence of fine new blood vessels in gastrocnemius and soleus muscle of transplanted limb, and improvement or recovery of blood vessel contrast in anterior and posterior tibial artery and pedal artery are observed after the transplantation.
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Repetitive bypass grafting surgery was performed, but gave him only insufficient result. Thus, we decided to perform therapeutic angiogenesis for his left limb. Angiography indicated obstruction of posterior tibial artery and dorsal pedal artery in his left limb. As a result of the operation, in total, 1.4 × 109 BMMNCs were harvested and implanted into his left limb at 60 points. There was no remarkable complication or adverse symptom due to the transplantation. Two weeks after the operation, numbness and leg pain were reduced. The findings of thermography revealed that augmentation of blood flow in patient's treated leg was suggested by elevation of skin temperature at 1 week after of the treatment. 3.3. EPC assay In the first and second cases, the frequencies of CFU-EC were 26.8 and 18.5/106 BMMNCs, respectively (Fig. 3A). These were almost equivalent results to those of healthy adult BM (19.0 ± 10.0/106 BMMNCs). Furthermore, FACS analysis revealed that frequency of EPCs in marrow graft of the first and second cases were 0.1% and 0.039%, respectively. These results were also equivalent to those of healthy adult BM (0.057± 0.016%). 3.4. Growth factors We investigated changes of serum level of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) which were both considered to play an important role
Fig. 3. Findings of EPC assay and cytokines. (A) A picture of representative colony forming unit-endothelial cell (CFU-EC) in Case 2. (B) Changes in serum level of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). VEGF and HGF were quantified by EIA and ELISA, respectively.
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in neovascularization. In both cases, serum level of VEGF was markedly elevated in posttransplantation phase (Fig. 3 B). 4. Discussion In the present study, we recognized the effectiveness and safety of therapeutic angiogenesis by implantation of autologous BMMNCs as indicated in the successful report from TACT trial [4]. Accumulation of clinical cases and large-scale clinical studies are necessary to clarify long-term verification of safety and effectiveness. Furthermore, we should be aware that there are some controversies in this procedure. The discovery of EPCs has drastically changed our understanding of adult blood vessel formation. The finding that EPCs home to sites of neovascularization and differentiate into ECs in situ is consistent with “vasuculogenesis,” a critical paradigm well described for embryonic neovascularization. On the other hand, the so-called “angiogenesis” are caused by activation of in situ ECs by various pro-angiogenic factors (i.e., VEGF, HGF, granulocyte-colony stimulating factor: G-CSF, platelet-derived growth factor-BB: PDGFBB, and matrix metalloproteinase 9: MMP-9, etc.). More recently, there are several studies that the therapeutic angiogenesis might function as an integrated therapy of in situ cells, cellular sources in transplanted BM, and pro-angiogenic factors, suggesting vascular restoration in treated limbs was achieved by collateral remodeling through paracrine systems [5]. Indeed, we could observe elevation of serum VEGF level in both cases in the post-transplant phase. Thus, in order to get the best of therapeutic efficacy, we should further investigate cellular and molecular mechanisms of the reconstruction of blood vessels, especially of arteriogenesis with maturation by acquisition of smooth muscle cell coat and extracellular matrix in physiological, pathological conditions, and in the process of utilizable therapeutic angiogenesis. Since there might be a danger of BM failure simultaneously in patients with radiation exposure, an availability of autologous marrow cells is limited in many exposured cases. Therefore, it is overwhelmingly likely that appropriately selected patients will undergo an allogeneic transplant procedure, although there are several barriers to allogeneic transplantation which limit its effectiveness, such as immunological effects between donor and recipient, and considerable time for donor search [6]. Furthermore, HSCT is presently recommended only for individuals who do not suffer significant injury to nonhematopoietic organs, since such multiple concomitant injuries to non-hematopoietic organs limit the effectiveness of HSCT [7]. On the other hand, in vivo evidence was recently reported which revealed neoendothelialization by donor-origin cells in a clinical case of the Tokai-mura nuclear accident which occurred in 1999 [8]. Therefore, our challenges in this field should include exploration of novel approaches which could expand the role of cell transplantation therapy in the victims of radiological events from reconstruction of hematopoietic function to restoration of non-hematopoietic organs. We should develop novel cellular sources and the best way of isolation and ex vivo expansion of stem cells and regulation of their survival and function following transplantation with the combined use of soluble factors and/or bioengineering techniques. Exploration of such new strategies might shed light on new directions for tissue regeneration medicine for victims of radiation exposure.
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References [1] K.L. Koenig, R.E. Goans, R.J. Hatchett, et al., Medical treatment of radiological casualties: current concepts, Ann. Emerg. Med. 45 (2005) 643–652. [2] L.F. Fajardo, M. Berthrong, Vascular lesions following radiation, Pathol. Annu. 23 (1988) 297–330. [3] T. Asahara, T. Murohara, A. Sullivan, et al., Isolation of putative progenitor endothelial cells for angiogenesis, Science 275 (1997) 964–967. [4] E. Tateishi-Yuyama, H. Matsubara, T. Murohara, et al., Therapeutic Angiogenesis Using Cell Transplantation (TACT) Study Investigators, Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomized controlled trial, Lancet 360 (2002) 427–435. [5] T. Kinnaird, E. Stabile, M.S. Burnett, et al., Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms, Circ. Res. 94 (2004) 678–685. [6] N. Dainiak, R.C. Ricks, The evolving role of haematopoietic cell transplantation in radiation injury: potentials and limitations, Br. J. Radiol., Suppl. 27 (2005) 169–174. [7] J.K. Waselenko, T.J. MacVittie, W.F. Blakely, et al., Strategic National Stockpile Radiation Working Group, Medical management of the acute radiation syndrome: recommendations of the Strategic National Stockpile Radiation Working Group, Ann. Intern. Med. 140 (2004) 1037–1051. [8] T. Suzuki, M. Nishida, S. Futami, et al., Neoendothelialization after peripheral blood stem cell transplantation in humans: a case report of a Tokaimura nuclear accident victim, Cardiovasc. Res. 58 (2003) 487–492.